Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

Zhiyun Wei; Arsen Batagov; Sergio Schinelli; Jintu Wang; Yang Wang; Rachid El Fatimy; Rosalia Rabinovsky; Leonora Balaj; Clark Chen; Fred Hochberg; Bob Carter; Xandra Breakefield; Anna Krichevsky

doi:10.1038/s41467-017-01196-x

Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

Wei, Zhiyun; Batagov, Arsen; Schinelli, Sergio; Wang, Jintu; Wang, Yang; El Fatimy, Rachid; Rabinovsky, Rosalia; Balaj, Leonora; Chen, Clark; Hochberg, Fred; Carter, Bob; Breakefield, Xandra; Krichevsky, Anna 2017-10-26 00:00:00 ARTICLE DOI: 10.1038/s41467-017-01196-x OPEN Coding and noncoding landscape of extracellular RNA released by human glioma stem cells 1 2 3 4 1 1 Zhiyun Wei , Arsen O. Batagov , Sergio Schinelli , Jintu Wang , Yang Wang , Rachid El Fatimy , 1 5 6 7,8 7 5 Rosalia Rabinovsky , Leonora Balaj , Clark C. Chen , Fred Hochberg , Bob Carter , Xandra O. Breakeﬁeld & Anna M. Krichevsky Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reﬂecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal ﬂuid. Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, HMS Initiative for RNA Medicine, Boston, MA 02115, USA. 2 3 Vishuo Biomedical, #3-33 Teletech Park, 20 Science Park Road, Singapore 117674, Singapore. Department of Drug Sciences, University of Pavia, Pavia 4 5 27100, Italy. Beijing Genomics Institute, Shenzhen 518083, China. Department of Neurology and Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Charlestown, MA 02129, USA. Neurosurgery Department, University of Minnesota, Minneapolis, MN 55455, 7 8 USA. Department of Neurosurgery, University of California, La Jolla, San Diego, CA 92093, USA. Scintillon Institute, San Diego, CA 92121, USA. Correspondence and requests for materials should be addressed to A.M.K. (email: [email protected]) NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 1 | | | 1234567890 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ntercellular communication within complex biological sys- investigation of cancer-derived exRNA. As proliferating and tems, such as cancer and its host microenvironment, via invading GBM cells migrate through brain parenchyma, they I“horizontal” RNA transfer, is an expanding area of research . interact with the changing landscape of extra-tumoral stimuli and Extracellular RNAs (exRNAs) are packaged into various extra- actively modulate this landscape . Communication between cellular complexes, including microvesicles (MVs), exosomes, and tumor cells and diverse normal cells in the brain is nevertheless 2, 3 non-vesicular ribonucleoprotein complexes (RNPs) . MVs and one of the least investigated aspects of glioma biology. We exosomes, broadly called extracellular vesicles (EVs), are released employed low-passage patient-derived tumorigenic GBM cell and taken up by various cells, thereby transferring their content. cultures that represent the most therapy-resistant stem-like cell This process likely plays a role in cancer development and population (GSC), and are considered the core cell type within manipulation of its microenvironment . However, methodologies the tumor. Analysis of GSC cellular and extracellular RNA, along are only beginning to emerge for characterizing the exRNA with the transcriptome of primary human and mouse cells of the landscape and monitoring levels of individual coding and reg- brain microenvironment (neurons, astrocytes, endothelial cells, ulatory exRNAs. exRNA mostly consists of small RNA species and microglia) enables us to predict the most impactful miRNAs (<200 nt); and the majority of reports to date focus on and expand the repertoire of potentially transferred exRNAs far 5, 6 miRNA . As a critical step toward understanding the biological beyond the classes of miRNAs and mRNAs. We also demonstrate impact of exRNA release and transfer, we investigated the com- that MVs, large vesicles of 0.2–0.8 μm, most closely mirror the plete spectrum of cancer-derived exRNAs, and the enrichment of cellular transcriptome and thus present a highly promising but speciﬁc RNA classes and individual species. By creating cDNA yet poorly explored source of liquid biopsy biomarkers. libraries of both small and long exRNA, and reducing the ligation bias favoring miRNAs, we identiﬁed a diverse and highly distinct composition of exRNA in MVs, exosomes, and RNP complexes. Results Furthermore, semi-absolute quantiﬁcation of RNAseq, performed Sequential ﬁltration-based exRNA isolation. To characterize using RNA spike-in molecules, allowed us to monitor the levels of exRNA released by patient-derived GBM cells in various com- various RNA classes and species in these extracellular complexes. plexes, we assessed several technical approaches. EV and exRNA This work focused on glioblastoma (GBM), the most common isolation protocols can be generally categorized into three major and aggressive brain tumor, as an important model for groups: based on ultracentrifugation (UC), precipitation using ab d 0.22 μm 0.02 μm RNA sequencing pipeline RNA isolation pipeline 40–2000 ng RNA Cell Conditioned media rRNA depletion NS 300 g 10 min ** 25 NS UC 8–25% 92–75% 2000 g 15 min Filtration 0 0 SMARTer for long RNA TAP and T4PNK Add RNase inhibitor (with fragmentation) treatment 2.0 μm filtration NEBNext for small RNA HiSeq2000 SR50 c Advantages of filtration based exRNA isolation (select 15–65 nt insert) Sequential filtration 0.8 μm filtration superior to: Benefits rRNA mapping HiSeq2000 SR50 MV Clearer separation 0.22 μm filtration >100 folds lower pressure on EV mRNA mapping rRNA mapping Higher yield Exosome 0.02 μm filtration No external chemical Less variation between batches miRNA mapping ncRNA mapping Require no capital equipment RNP 3 kDa concentration Applicable to big volume Abbreviations: tRNA, Y RNA, Rfam RNA isolation with UC: Ultracentrifugation PP: Polymer precipitation and other mappings DNase treatment DG: Density gradient UC GF: Gel filtration Fig. 1 Flowchart of the exRNA fractionation and sequencing. a The pipeline of the ﬁltration-based exRNA isolation. Following removal of cells and cellular debris by low speed centrifugation, the supernatants were ﬁltered through a sequence of reduced pore sizes (2.0, 0.8, 0.22, and 0.02 μm) to separate the extracellular fractions, and a ﬁnal concentrator with the cutoff of 3 kDa was applied to collect the remaining small particles. b The aliquots of conditioned media after 0.8 μm ﬁltration were used for MV and exosome isolation, either by ultracentrifugation (UC) or ﬁltration, and the RNA yield of these fractions compared. The number of remaining vesicles/particles was compared in UC supernatant and ﬁlter ﬂow-through. N = 4 aliquots of conditioned media. All bars represent mean ± SEM. c Comparison between the ﬁltration-based exRNA isolation and other common exRNA isolation methods. The stars mark superior characteristics of sequential ﬁltration over other methods. d The optimized pipeline for the broad coverage, minimally biased RNA-sequencing. RNA of 15–65 nt was selected for the small RNA libraries, to reduce the overwhelming levels of tRNAs. NS, not signiﬁcant; *p < 0.05; **p < 0.01; t-test 2 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | Isolated MV Isolated exosome After MV isolation After exosome isolation RNA concentration (ng/ml media) 0.8 μm filtrate 16,000 g 0.5 h Vesicle/particle number (*10 per ml) 100,000 g 2.5 h NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE ab Ladder (kDa) Cell MV Exosome RNP MV Exosome RNP 52- Flotillin-1 26- CD9 ANG (angiogenin) 10- Integrin β1 140 - 0.5 μm 0.1 μm 0.1 μm 95- HSP90 52- La antigen MGG75 GBM8 NPM GBM4 34- (nucleophosmin) 20/3 Ago2 95- 5 (argonaute 2) 140 - Alix 95- PCNA 34- (proliferating cell 26- nuclear antigen) 72- Ro60 (TROVE2) 52- Fig. 2 Quality control of the fraction separation. a Transmission electron microscopy of EVs and RNPs isolated using the sequential ﬁltration protocol. TEMs were replicated three times. b Protein markers, identiﬁed by the western blotting, veriﬁed the separation of extracellular fractions. Equal protein input of 50 μg per lane was used. Western blots were replicated twice. c RNA yields of extracellular fractions from different GSC cultures chemical polymers (PP), such as polyethylene glycol, and frac- different patients varied in the amount of exRNA released tionation, including density gradient UC and gel ﬁltration (Fig. 2c), ranging between 5.4 and 38.0 ng/ml accumulated in (DG&GF) . Since speciﬁc markers or physical parameters for the culture over 7 days. Considering that 3–10 μg of cellular RNA was various types of EVs and extracellular RNPs are still not clearly isolated from 1 ml of the corresponding GSC neurosphere deﬁned, UC remains the most commonly used approach to iso- cultures (~1.3 million cells), between 0.05 and 0.7% of cellular late the entire vesiculome . However, based on nanoparticle RNA is accumulated in the extracellular space in 7 days. Of note, tracking analysis (NTA; NanoSight) and ﬂuorescent dye-binding total exRNA yield varied ~7-fold among the GSC cultures, and quantiﬁcation (RiboGreen), the yield of EVs and exRNA isolated the proportion of exRNA associated with different extracellular by this technique is relatively low (20–40%) (Supplementary complexes also varied between the GSC types, suggesting the Fig. 1). Furthermore, this procedure yields a highly heterogeneous variations between cultures reﬂected intrinsic properties of 9, 10 mix of EVs and RNP/LNP (liponucleoprotein) particles .To different tumors. Analytical RNA proﬁles examined by the separate EVs and RNPs according to their physical size and Agilent 2100 Bioanalyzer indicated high-quality cellular RNA improve the yield of exRNA isolation, we developed a sequential (RIN > 9.5) with sharp rRNA peaks and no sign of degradation ﬁltration (SF) protocol (Fig. 1a). This protocol offers several (Supplementary Fig. 3). In contrast, exRNA exhibited mostly advantages over current methods, including low pressure on EVs, short RNA proﬁles (below 200 nt) with intact rRNA peaks better separation between EVs and RNPs, higher RNA yield, and detectable in large MVs, but not exosomes and RNPs. scalability (summarized in Fig. 1b, c and Supplementary Fig. 1). However, the extended hands-on time of the ﬁltration procedure (Supplementary Fig. 2) and the separation solely on the basis of Technical considerations for RNAseq. The protocols commonly size are limitations of this method. Also, retrieval of EVs from the utilized for small RNA library construction are based on adaptor ﬁltration membranes is inefﬁcient and could potentially alter their ligations to the 5′-phosphate and 3′-hydroxy ends of RNA, the structure; therefore, the utility of this protocol for functional EV modiﬁcations characteristic for miRNA, and thus favor analysis needs additional evaluation. miRNA . In order to characterize RNA content in a minimally Established glioma cell lines have very limited capacity to biased way, we utilized sequential treatments with tobacco acid reﬂect GBM biology . We utilized previously characterized low- pyrophosphatase (TAP) and T4 polynucleotide kinase (T4 PNK) passage GSC cultures derived from four primary human to create more uniform 5′ and 3′ ends for various types of 12, 13 heterogeneous GBM tumors for the exRNA proﬁling . These transcripts, leading to their more accurate representation in the 16, 17 cells were grown as neurospheres in serum-free medium, to cDNA libraries . The caveat is that this end-modifying pro- maintain their initial properties and transcriptional proﬁles and, cedure leads to an overwhelming abundance of rRNA reads in therefore, better reﬂect tumor biology . Transmission electron cellular and exRNA samples, and reduces the sequencing depth microscopy conﬁrmed the presence of EVs/particles in the for other RNA classes. Therefore, we included an rRNA depletion corresponding extracellular fractions isolated from GSC cultures step in the protocol that reduced rRNA reads remarkably (Fig. 2a). Distinct proﬁles of several protein markers exhibited by (Fig. 3a). cellular and extracellular fractions served to conﬁrm the purity of Unlike the established strategies for normalization of cellular fractions and the lack of cellular contamination in the MV and RNAseq data sets, which utilize total mapped reads as the other extracellular fractions (Fig. 2b). GSC cultures derived from normalization factor, there are no adequate standard for NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 3 | | | MV Exosome RNP RNA concentration (ng/ml media) RNP-enriched Exosome-enriched MV-enriched ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x a b 14 Without rRNA depletion With rRNA depletion Conditioned media Long RNA library Small RNA library Long RNA library Small RNA library Fresh media 9.7% 27.4% 8 MV MV MV MV long RNA small RNA long RNA small RNA library library library library 89.6% 85.7% 2 MV Exosome RNP 9.5% 36.7% Exosome Exosome Exosome Exosome long RNA small RNA small RNA c long RNA library library library library 92.5% 90.5% MV Exosome RNP 33.5% RNP RNP RNP RNP long RNA small RNA long RNA small RNA library library library library 92.8% 63.9% Before After rRNA reads Non-rRNA reads correction correction Fig. 3 rRNA depletion and media correction are warranted for the deep RNAseq analysis. a Comparison of the exRNA libraries prepared with and without rRNA depletion. The percent of rRNA reads is shown, indicating that the majority of exRNA reads in non-depleted libraries represent rRNA. Sequencing depth of other exRNA classes is substantially increased by the rRNA depletion. b Quantiﬁcation of total RNA in conditioned and fresh media indicates that the vast majority is cell derived (n = 4 GSC cultures). Nevertheless, quantiﬁcation of speciﬁc RNA species can be skewed by the media, as illustrated in c. The levels of miR-122 were assessed in exRNA isolated from the fractions of conditioned and fresh media. miR-122 enrichment in exRNA (n = 4 GSC cultures) was calculated pre- and post-correction to the levels in fresh media, as described in Supplementary Fig. 6. miR-122 was highly enriched in the GSC exRNA, relative to its intracellular level, with up to 1500-fold enrichment in the exosomes before media correction. miR-122 enrichment in GSC exosomes became marginal upon correction. All bars represent mean ± SEM comparative quantitative assessment of cellular vs extracellular mRNA or non-coding RNA. Although in three out of four GSC RNAs. Since the proportion of total small RNA, and speciﬁc types cultures, cellular mRNA reads accounted for 20–35% of long of RNA is vastly different in cellular and extracellular RNA RNA libraries, the mRNA proportion in EV fractions was below (Fig. 4a and Supplementary Fig. 3), normalization to total 10%, and even lower in RNPs (Fig. 4a). The reads obtained from mapped reads is not optimal. As recently proposed , we utilized small RNA libraries were ﬁrst mapped to the most accurately spike-in RNA for normalization, and quantiﬁed the abundance of annotated miRNA database (miRBase), and subsequently to other RNA species as fmol per μg of total RNA. Further, since fresh databases with equal mapping priority. In total, all annotated culture media (FM) contain RNA that is co-isolated with cell- RNA species were categorized into 14 classes (Fig. 4a). Since small derived exRNA , we also assessed the interference of FM RNA RNA libraries were built on 15–65 nt transcripts, the vast with downstream analysis of GSC exRNA. With this goal, we majority of the mapped reads represent fragments rather than isolated three RNA fractions from the corresponding FM, using full-length transcripts, with exception of miRNA and piRNA the same ﬁltration procedure, and subjected them to RNAseq in reads. Despite the remarkable heterogeneity of the GSC cultures, parallel with the GSC exRNA. Approximately 1 ng/ml total different extracellular fractions exhibited common characteristics exRNA was isolated from FM, and it was largely associated with of their RNA repertoires. Some of the most distinct features of the RNP fraction (Supplementary Fig. 4). Overall, FM contributed GSC-derived exRNA are summarized as follows: (1) mRNA 1.3–15% to the exRNA isolated from conditioned media, varying exons and snoRNAs are depleted, compared to cellular RNA, in between the fractions (Fig. 3b). Although this amount of all extracellular fractions; (2) all extracellular fractions, especially contaminating RNA is small, it can still affect the results of non-vesicular RNPs, are highly enriched in speciﬁc Y RNA exRNA enrichment analysis. For example, miR-122 falsely fragments of largely unknown functions; (3) MVs and exosomes showed exosomal enrichment (Fig. 3c), consistent with previous differ in their RNA composition, with mRNAs being relatively 20, 21 reports , due to its abundance in B-27 supplement more enriched in MVs and miRNAs in exosomes; (4) RNP (Supplementary Fig. 5). Based on this observation, we included fractions have a highly distinctive RNA repertoire, with tRNA FM RNA data set in our RNAseq analysis pipeline, to provide the and Y RNA fragments strongly enriched, and snRNA and repeats baseline for GSC-derived exRNA (Supplementary Fig. 6). The reduced. The predominance of tRNA and Y RNA fragments in results described below were obtained using the media correction. RNP is reﬂected in the corresponding sharp ~32 nt peak observed in the length distribution proﬁle of reads (Supplementary Fig. 7). The relative abundance of piRNA and scRNA (small cytoplasmic EVs and RNPs exhibit distinct RNA composition. To char- RNA) fragments was also higher in RNP fractions. However, the most abundant individual piRNA and scRNA species were acterize the repertoire of GSC cellular and extracellular RNA, we sequenced the libraries of small and long RNAs, and ﬁrst nor- identical or highly homologous to major tRNA and Y RNA fragments, respectively (Supplementary Fig. 8). Whether such malized the number of reads for each RNA class to the total identical sequences indeed belong to two functionally distinct number of non-rRNA reads within the library, thereby removing the confounding factor of variable rRNA depletion efﬁciencies. classes of transcripts, or tRNA and Y RNA are commonly mis- identiﬁed due to the poor quality of databases, is unknown. In All reads generated on the long RNA libraries (that generally included transcripts longer than 100 nt), were classiﬁed as either addition, recently discovered circular RNAs have been reported as 4 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | RNP Exosome MV 4.0% miR-122 enrichment (ratio to cellular RNA) RNA concentration (ng/ml media) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE Cell MV Exosome RNP Long RNA: 50 ncRNA 40 mRNA Small RNA: miRNA tRNA Y RNA 70 piRNA snRNA snoRNA SRP RNA scRNA 40 vRNA mRNA exons mRNA introns Repeat Other ncRNA 10 Other genome bd ** *** *** mRNA Full length Amplicon length NS NS NS NS * * 0.5 RPS27 361 nt 306 nt RPLP2 511 nt 451 nt 0.0 RPS6 829 nt 724 nt –0.5 EEF2 3163 nt 2996 nt 3′-UTR 5′-UTR YY1AP1 2705 nt 2509 nt CDS –1.0 MV Exosome RNP PROM1 3857 nt 3708 nt Cell MV Exosome RNP >6000 5500–6000 5000–5500 20/3 4500–5000 GBM4 4000–4500 GBM8 3500–4000 MGG75 3000–3500 2500–3000 2000–2500 1500–2000 1000–1500 500–1000 0–500 Number of transcripts Fig. 4 Relative composition of diverse RNA classes in cellular and extracellular compartments (MVs, exosomes, and RNPs) in different GSC cultures. a The top panels exhibit relative RNA composition in long RNA libraries, and the bottom panels depict the composition in small RNA libraries. The data were normalized to the total number of annotated non-rRNA reads. The bars framed in red represent the mean values of four GSC cultures. b RT-PCR analysis (with equal input of total RNA) of selected mRNAs abundant in exRNA, demonstrates the presence of nearly full-length short, but not long messages in the extracellular fractions. Long RT-PCRs were replicated twice. c Long RNA libraries-based analysis of the length distribution of 500 most abundant mRNAs suggests no length preference for shorter parent transcripts in the extracellular fractions. d Analysis of the RNAseq reads mapped to mRNAs indicates that UTR regions were more enriched than CDS regions in the extracellular fractions (n = 4 GSC cultures). mRNA reads were aligned to the coding sequences (CDS), 5′-UTRs, and 3′-UTRs separately, and the alignment rates for each extracellular fraction were normalized to the corresponding cellular fraction. The log-transformed ratios of the alignment rates were compared among the three regions. Error bars represent mean ± SEM. NS, not signiﬁcant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test 22 24, 25 enriched in exRNA , and although our RNAseq protocol was not fragments in exRNA . On average, our RNAseq detected optimized for their quantiﬁcation, several circular RNA species 17,148 intracellular mRNAs in GSCs, whereas 17,219, 11,592, and were detected (Supplementary Table 1). 11,819 mRNAs were detected in MV, exosome, and RNP frac- tions, respectively. Many of the most abundant extracellular mRNAs corresponded to relatively short transcripts, including UTR fragments are released more than ORFs and intact mRNAs for various ribosomal proteins (Supplementary Data 1). mRNAs. The length of intact cellular mRNAs varies between 350 However, some abundant reads in EVs corresponded to long and 12,000 nt, with the average around 2000 nt . Considering the mRNA transcripts, such as PROM1 and EEF2. To examine small size of EVs, the maximum length of an mRNA that could be whether intact mRNAs are present in extracellular fractions, we packaged is still an open question. Previous high-throughput designed PCR primers for selected mRNAs to provide their full- studies have not discriminated between full-length mRNAs and length ampliﬁcation. As shown in Fig. 4b, near-complete short NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 5 | | | 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 20/3 GBM4 GBM8 MGG75 Mean Cell 20/3 MV GBM4 Exosome GBM8 RNP MGG75 Mean 20/3 GBM4 GBM8 MGG75 Mean 20/3 GBM4 GBM8 MGG75 Mean Relative abundance (%) Length range (nt) Log ratio of alignment rate to cell ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x Evenness factors of RNA species abundance distribution RNA category Cell MV Exosome RNP All long RNA 6.37±1.49 4.71±1.53 4.67±1.04 4.92±0.47 --mRNA 11.70±0.89 12.12±0.36 14.27±3.85 16.30±2.45 --ncRNA 2.59±0.72 4.26±0.89 2.89±1.76 3.81±0.40 All small RNA 3.94±0.26 3.12±0.10* 2.26±0.19** 0.91±0.15*** --miRNA 6.09±0.34 5.27±0.12 5.39±0.35 4.76±0.30* --snoRNA 16.62±1.79 17.98±0.72 16.24±1.27 9.41±2.57 11.63±0.57 8.10±0.69** 6.98±0.58** 5.99±0.99** --snRNA --SRP RNA 17.81±0.79 15.57±0.45* 12.32±1.05** 14.19±1.43 --tRNA 19.20±0.45 17.41±0.45* 11.61±0.89*** 8.48±0.45*** --vRNA 31.25±6.25 31.25±6.25 25.00±0.00 18.75±6.25 --Y RNA 11.27±0.98 8.33±0.56* 6.54±0.27** 5.88±0.38** b c Fold change of X value to estimate heterogeneity difference Cell MV GBM4 GBM8 GBM4 GBM8 RNA category Cell MV Exosome RNP 15 17 17 17 All long RNA 1 1.340 1.720 1.660 8 10 4 2 71 --mRNA 1 0.967 2.746 2.905 18 88 26 19 14 8 29 --ncRNA 1 1.792 2.137 1.998 46 45 4 0 7 4 All small RNA 1 1.868 2.651 1.624 3 5 1 3 8 9 20/3 MGG75 20/3 MGG75 --miRNA 1 1.045 0.838 1.001 Exosome RNP --snoRNA 1 2.034 1.762 2.196 GBM4 GBM8 GBM4 GBM8 --snRNA 1 2.060 1.995 4.266 54 36 76 79 --SRP RNA 1 1.073 3.520 3.069 2 615 10 41 50 611 35 67 80 89 --tRNA 1 0.423 0.916 1.889 15 1 --vRNA 1 0.692 0.068 0.958 1 5 0 5 1 7 2 11 6 --Y RNA 1 0.401 1.030 1.002 20/3 MGG75 20/3 MGG75 Fig. 5 Inequality and heterogeneity of the RNA repertoire of extracellular fractions. a Evenness factors, reﬂecting the inequality of abundance’ distribution of the indicated RNA classes in various fractions. Higher evenness factors correspond to lower inequality. The classes of tRNA, Y RNA, snRNA, and SRP RNA, but not miRNA, contributed to the increased inequality of exRNA most signiﬁcantly. Differential evenness factors of cellular and extracellular RNA suggest the selectivity of secretion (n = 4 GSC cultures). b For each RNA category, a sum of squared errors (χ value) was calculated among four GBM cultures, after normalization of each RNA species to the total number of reads in that RNA category. The χ value of each extracellular fraction was compared to the cellular fraction. Fold change of χ values higher than 1 reﬂects the increased heterogeneity. Heterogeneity either increased or decreased more than two-fold is highlighted in red and blue, respectively. c Venn diagrams depict the number of common species among 100 top abundant mRNAs, in four GBM cultures and their extracellular fractions, supporting the observation of higher heterogeneity of mRNA composition in exRNA than in cellular RNA. Signiﬁcant differences between the cellular and extracellular fractions are depicted as following: *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test mRNAs could be detected in cellular and all extracellular frac- usually account for the majority of total miRNA in a given cel- tions, but detection of long mRNAs above 1000 nt was limited to lular context . Consistent with this observation, 31 miRNA cells and MVs only. These results suggest that either long mRNAs species accounted for 80% of the total miRNA in GBM8 cells, are excluded from packaging into exosomes and RNP complexes, indicative of a diverse range of miRNA expression (or “inequal- or they are present in these fractions only as fragments. The latter ity” of miRNA levels). Even fewer, 19 miRNA species, accounted appears to be the case, since the RNAseq demonstrates a similar for 80% of the total miRNome in GBM8 exosomes, suggesting a representation of transcripts of various lengths in the intracellular higher inequality of miRNAs in exRNA. Such comparison, and extracellular compartments (Fig. 4c). These data suggest that however, relies on a randomly selected cutoff (80% used above). most exRNA reads corresponding to long mRNAs represent To compare the inequality of intracellular and extracellular fragmented transcripts. Nevertheless, amplifying long RNA from transcripts more objectively, we developed two alternative stra- low-input exRNA fractions is technically challenging and cannot tegies. The ﬁrst one is an improved version of traditional eva- be performed in a high-throughput manner, so we cannot exclude luation named the evenness factor (ε), which deﬁnes that ε%of that some long full-length mRNAs are present extracellularly. RNA species can account for (100−ε)% of total abundance. The Next, mRNA reads were aligned separately to the coding second strategy is based on the Gini coefﬁcient, commonly used sequences (CDS), 5′-UTRs, and 3′-UTRs. The UTR regions, and for evaluation of inequality in economics. Of note, although both especially 3′-UTRs, were signiﬁcantly enriched in all extracellular are objective and use no preset cutoff, higher ε and lower Gini fractions relative to CDS sequences (Fig. 4d), validating the pre- coefﬁcient correspond to a more equal representation (or lower vious observation and suggesting differential release pathways inequality). The detailed illustration of the concepts behind these for the protein-coding and regulatory sequences. evaluation systems can be found in Supplementary Fig. 9. Taking advantage of these parameters, we compared transcript inequality in cellular and extracellular fractions, either for the whole RNA Inequality of RNA representation increases in exRNA.A library or a speciﬁc RNA class. The inequality of RNA levels was relatively small number of the most abundant miRNA species similar for cellular and extracellular long RNA libraries, but 6 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | fmol input in RT fmol input in RT fmol input in RT NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE ab c Cell 15,000 50,000 RNY1 fragment GluCTC fragment 40,000 RNY3 fragment GlyGCC fragment 10,000 30,000 RNY4 fragment GlnTTG fragment 20,000 RNY5 fragment LeuCAA fragment 5000 MV 10,000 200 250 Exosome RNP 0 0 0 30 112 5′ 3′ Nucleotide position de f Y1 Y3 Y4 U2 - miR1246 –10 Y5 Y4 - 5′Y4 Y5 - 5′Y5 Glu - 5′Glu Gly - 5′Gly –2 –20 5′ 3′ miR-199a-3p miR-92a-3p 5′Y5 –4 –5 4 25 2 25 2 18 2 19 2 –6 26 2 –5 20 2 26 2 21 2 –7 2 0 22 2 –6 27 2 –1 23 2 –8 28 2 –2 24 2 –7 2 –3 –9 2 RNAseq 29 RNAseq 2 RNAseq –4 qPCR qPCR 26 qPCR –8 –10 –5 29 2 30 2 2 Cell MV Exosome RNP Cell MV Exosome RNP Cell MV Exosome RNP Fig. 6 Y RNA and tRNA fragments are abundant and enriched in exRNA. a, b Speciﬁc Y RNA and tRNA species are among the most abundant in small RNA libraries (n = 4 GSC cultures). The reads corresponding to Y1, Y4, and Y5 are highly enriched in extracellular fractions, especially in RNP (a). The reads corresponding to speciﬁc tRNAs, such as GluCTC and GlyGCC, are highly enriched in exRNA, while others (e.g. GlnTTG and LeuCAA) are not (b). c Mapping coverage of Y1 reads indicates that the Y1 is precisely processed, and mostly its 5′ fragment is present in exosomes and RNPs, as evidenced by the steep peaks corresponding to the 5′-end 30 nt. These proﬁles are distinct from the more uniform full-length coverage observed for the cellular RNA, and to a lesser extent MV RNA. Similar analysis for other Y RNAs and tRNAs is presented in Supplementary Fig. 12. d The predicted secondary structure of Y1 RNA , Copyright (1993) National Academy of Sciences, USA, and the position of its cleavage (indicated by the arrow) that produces the 5′ fragment which is highly abundant in exRNA. e Quantiﬁcation of Y RNA reads in long and small RNA libraries, demonstrates different fragment to full-length ratios in the cell and exRNA fractions (n = 3 GSC cultures; MGG75 was excluded due to very low abundance of the full-length Y RNA). The ratios are increased in extracellular fractions. f qRT-PCR analysis with primers speciﬁc to either full-lengths or fragments of several RNA species validates the enrichment of 5′ tRNA and Y RNA fragments in extracellular fractions. For each speciﬁc transcript examined, two lines represent GBM8 and 20/3 cells, respectively. g qRT- PCR analysis of selected transcripts conﬁrms the quantitative character of the RNAseq pipeline. The blue dots represent qRT-PCR Cq values, while the red dots represent the results of RNAseq quantiﬁcation in fmol. Both analyses were performed on the same set of RNA samples. Error bars represent mean ± SEM increased signiﬁcantly in small exRNA libraries (Fig. 5a and cellular and extracellular RNA species across the GSC cultures, we Supplementary Table 2). These results provide support for the ﬁrst normalized the reads to the total read number within an idea of selective RNA incorporation into different exRNA frac- individual RNA category, and then evaluated the sum of squared 2 2 tions, that is still highly debated in the ﬁeld. errors (χ value). The higher χ value reﬂects the higher diversity/ heterogeneity of GSC cultures in terms of RNA composition. As shown in Fig. 5b, relative to cellular RNA, the extracellular Heterogeneity of RNA repertoire increases in exRNA fractions. fractions were, overall, more heterogeneous in their composition Despite the genetic diversity of GSCs, cellular RNA class com- in both long and small RNA libraries, as well as for the majority position was similar among the four different GSC cultures of speciﬁc RNA classes. This phenomenon was not caused by analyzed, with the exception of MGG75 cells that expressed more technical irreproducibility of the exRNA analysis, because inde- mRNA than long ncRNA (Fig. 4a). However, the RNA repertoire pendently analyzed exosomes produced by different passages of of extracellular fractions was more heterogeneous among GSC GBM8 cells were much more concordant than the pairwise- cultures than that of cells. To estimate the heterogeneity of compared exosomes released by GSC cultures established from NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 7 | | | C el l MV Ex o s o m e RN P Ce l MV Ex o s o m e RN P Cell MV Exosome RNP Cell MV Exosome RNP Cq value Abundance (fmol/μg total RNA) Log ratio between fragment and full length Cq value Abundance (fmol/μg total RNA) Cq - Cq full-length fragment Cq value Prefer full-length Prefer fragment Sequencing depth ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x a cd ef All mRNAs: All miRNAs: 0.0 3.5 25.0 –4.0 –1.0 1.5 0.0 0.04 0.15 R =0.4954 0.1 0.01 0.1 0.001 piRNA 0.01 R =0.9169 tRNA 0.0001 0.001 Y RNA 0.0001 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 scRNA GBM4 cell GBM4 cell miRNA abundance+0.0001 abundance+0.001 vRNA snoRNA R =0.1129 mRNA exons 0.1 SRP RNA 10 snRNA 0.01 Other ncRNA 0.1 Other genome 0.001 R =0.1685 0.01 Repeat mRNA introns 0.0001 0.001 0.0001 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 GBM4 cell GBM4 cell abundance+0.0001 abundance+0.001 PCA analysis 1 1000 MV 2 2 R =0.0020 100 R =0.0260 Exosome 0.1 0.01 2 0 RNP 0.1 –1 0.001 0.01 –2 PC1 (46.7%) 0.001 0.0001 –4 –2 –2 0 –2 0.0001 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 –1 –1 –1 GBM4 cell GBM4 cell –2 abundance+0.0001 abundance+0.001 1 Cellular 1 –4 –2 0 2 * *** *** *** * * *** *** ** ** * * * *** *** ** ** ** *** ** *** * *** *** *** MV Exosome RNP COL1A2 PTEN EEF2 RPS6 miR-21 miR-10b GluCTC GlyGCC Y4 5′Y4 Y5 5′Y5 Fig. 7 RNA repertoire of MV most closely reﬂects cellular RNA composition. a Heat map cluster analysis of RNA classes indicates relative similarity of the composition of MVs and the source cells. The scale bar represents the percentage of non-rRNA annotated reads. b PCA analysis of RNA classes. Different fractions are marked in different colors. Within each fraction, four dots represent four GSC cultures. c, d GBM4 MV represents the cellular mRNA content closely, and much better than exosomes or RNPs, based on the correlation analysis of all mRNA species (c), and cluster analysis of the top abundant mRNAs (d). The scale bar represents mRNA abundance. e, f Extracellular miRNA composition, in general, is less reﬂective of the cellular miRNome; nevertheless, MV fraction still remains the best simulator, based on the correlation analysis of all miRNA species (e), and cluster analysis of the top abundant miRNAs (f). The scale bar represents the log-transformed miRNA abundance. Similar analyses of other cell cultures can be found in Supplementary Figs. 14–17. g qRT-PCR analysis of exRNA fractions isolated from the CSF of GBM patients, using the same ﬁltration-based procedure, indicates its applicability to clinical samples. The data verify preferential association of selected RNA species with different exRNA fractions in the human bioﬂuid (n = 4 CSF samples). All bars represent mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test different patients (Supplementary Fig. 10). Consistently, Venn tRNA species are highly abundant in extracellular fractions, diagram analysis showed less commonalities (or increased het- especially the RNP, reaching the quantities of up to dozens of erogeneity) for mRNAs in exosomes and RNPs, relative to cellular pmol per μg of total RNA (Fig. 6a, b). Relative to let-7b-5p, one of RNA (Fig. 5c). The heterogeneity of miRNA class, however, was the most abundant miRNAs in exRNA, those molecules are at similar between GSC cellular and exRNA compartments (Sup- least hundred times more abundant. Since human Y RNAs are plementary Fig. 11). Combined analyses of inequality and het- 84–113 nt and tRNAs are 68–176 nt, and our libraries were erogeneity suggest that GSC cultures may utilize various sorting constructed from 15 to 65 nt RNA, we reasoned that the reads mechanisms for exRNA release. represent fragments rather than full-length transcripts. Notably, the coverage analysis of the reads mapped to Y RNAs and tRNAs suggested the presence of speciﬁcally processed fragments (Fig. 6c and Supplementary Fig. 12), with the processing sites located Y RNA and tRNA fragments are abundant and enriched in within the loop domains that are known to bind several pro- exRNA. The reads corresponding to tRNA and Y RNA species 28–30 teins (Fig. 6d and Supplementary Fig. 12). Further integra- constitute a signiﬁcant proportion of the rRNA-depleted small tion of the small and long RNA data sets revealed that the ratios exRNA libraries (8.5% in cells, 13.5% in EVs, and 67.5% in RNPs, of fragment to full-length Y RNAs differed signiﬁcantly among Fig. 4a). All four human Y RNA (Y1, Y3, Y4, and Y5) and some 8 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | PC3 (13.0%) Cell PC2 (17.7%) % of total exRNA MV from CSF Exosome RNP GBM4 RNP GBM4 exosome GBM4 MV abundance+0.0001 abundance+0.0001 abundance+0.0001 Top abundant mRNAs: Cell MV Exo RNP GBM4 RNP GBM4 exosome GBM4 MV abundance+0.001 abundance+0.001 abundance+0.001 Top abundant miRNAs: Cell MV Exo RNP Percentage NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE GBM MV vs HA GBM exosome vs HA GBM RNP vs HA –5 –4 –5 –4 –4 miR-196a-5p –3 miR-196a-5p miR-196a-5p miR-423-5p miR-615-3p –3 –3 miR-615-3p miR-378a-3p miR-10b-5p miR-10b-5p –2 miR-615-3p miR-378a-3p miR-10b-5p miR-378a-3p –2 –2 miR-182-5p miR-183-5p miR-182-5p miR-182-5p miR-183-5p –1 miR-199a/b-3p –1 –1 miR-378c –2 –1 0 1 2 3 –3 –2 –1 0 1 2 3 –4 –3 –2 –1 0 1 2 3 Mean log (FC) Mean log (FC) Mean log (FC) 10 10 10 Total in Total in miRNA hAst hNG HBEC mAst mMG mNeu MBEC TCGA GBM TCGA Ctrl p value human mouse miR-182-5p +++ – +++ 6 +++ +++ +++ – 9 7.823±0.064 6.789±0.054 0.024 miR-196a-5p +++ + + 5 NA NA NA NA NA 6.474±0.033 5.688±0.005 9.0E–04 miR-10b-5p +++ + – 4 +++ +++ +++ – 9 8.663±0.063 6.681±0.006 1.1E–05 miR-183-5p + – ++ 3 + +++ + – 5 6.497±0.037 6.024±0.010 0.078 miR-378a-3p +++ – –3 – – – – 0 5.929±0.004 5.908±0.011 0.524 miR-199a-3p + + – 2 – + + ++ 4 7.705±0.051 6.786±0.108 0.011 miR-199b-3p ++– 2 – + + + 3 NA NA NA miR-615-3p + + – 2 NA NA NA NA NA 6.098±0.019 5.886±0.018 0.127 miR-92b-3p – – + 1 – +++ – + 4 8.187±0.030 6.818±0.050 7.0E–10 miR-378c +–– 1 – – –– 0 NA NA NA Positive z-score z-score = 0 Negative z-score No activity pattern available Percentage 15.0 12.5 10.0 7.5 5.0 2.5 0.0 de 1.5 ** ** * * ** ** 1.0 0.5 40 Mono-culture Mono-culture Co-culture Co-culture 0.0 Timp3 Rassf9 Ptgfr Hmgb3 Hapln Cep110 U6 miR-21 Fig. 8 Comparative analysis of GSC-secreted miRNAs and cellular miRNome of the normal cells of the brain predicts the most impactful GBM miRNAs in tumor-to-microenvironment communication. a Extracellular fractions of GSC cultures were compared to primary human astrocytes (HA), based on the corresponding RNAseq data sets. The fold-changes in miRNA levels were log transformed and a t-test was applied to examine the signiﬁcance of difference. MiRNAs with log-fold changes higher than 1.7, which corresponded to 50 times higher levels in the GSC-derived exRNA fractions relative to the recipient cells, and p < 0.05 (t-test), were deﬁned as potentially impactful (colored in red). The horizontal axis of Volcano plots shows the log-fold difference, and the vertical axis shows the statistical signiﬁcance. Similar analyses of primary human neuroglial and endothelial cells are shown in Supplementary Fig. 18. b A full list of most impactful GSC miRNAs for human and mouse astrocytes, neurons, microglia, or brain endothelial cells. The number of “+” symbols reﬂects the number of extracellular fractions in which an miRNA meets the indicated criteria as in a. Most of these miRNAs are also upregulated in the GBM tumors compared to non-neoplastic brain tissues in the TCGA microarray data set, as indicated in the three right columns (n = 496 GBM vs 10 control). c Top enriched IPA pathways for the validated mRNA targets of the most impactful miRNAs. Predicted activation and inhibition of pathways are labeled as orange and blue bars, respectively. The yellow line shows the percent of genes in each pathway that are validated targets. d, e Co-cultured with GBM8 neurospheres, primary miR-21-null astrocytes exhibit steady miR-21 levels (d) and downregulation of validated miR-21 targets (e). Cq value of miR-21 in mono-cultures was deﬁned as 45 (undetectable expression). N = 4 wells in 24-well plate. All bars represent mean ± SEM. *, p < 0.05; **, p < 0.01; t-test NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 9 | | | Molecular mechanisms of cancer Glucocorticoid receptor signaling ERK/MAPK signaling AMPK signaling Breast cancer regulation by stathmin1 B-cell receptor signaling ERK 5 signaling Protein ubiquitination pathway Epithelial adherens junction signaling PI3K/AKT signaling Hypoxia signaling in the cardiovascular system NGF signaling GNRH signaling PPARα/RXRα activation HGF signaling Prostate cancer signaling CD27 signaling in lymphocytes Role of PKR in interferon induction and antiviral response Small cell lung cancer signaling Apoptosis signaling Cardiac hypertrophy signaling Regulation of elF4 and p7056K signaling CDK5 signaling Log (p) Cq value –Log(p-value) Log (p) Fold change (normalized to Actb) Log (p) 10 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x the Y RNA species (Fig. 6e). Overall, the fragment to full-length detected in CSF MVs. The preference for full-length Y RNAs in ratio was signiﬁcantly higher in extracellular vs cellular fractions CSF MVs, and their fragments in RNPs was also consistent with for all four Y RNA species (Fig. 6e), indicating that whereas full- the observations made on GSC-derived exRNA. length Y RNAs are highly abundant in the cells and MVs, speciﬁc fragments of them are enriched in the exosomes and RNPs. These Exosomal miRNAs with high functional potential upon data were further conﬁrmed by qRT-PCRs (Fig. 6f). Moreover, transfer. A common, but still highly debated, hypothesis is that such preference for fragments in the exosomes and RNPs was not EVs and EV-associated exRNA are taken up by recipient cells and limited to Y RNA. For example, full-length U2 snRNA (RNU2-1), mediate intercellular communication. MiRNA transferred from which also serves as a precursor for miR-1246 , and speciﬁc GBM to cells in their microenvironment could have signiﬁcant tRNAs are also released to a lesser extent in extracellular fractions impact on the transcriptome of the peritumoral cells. We rea- than their corresponding processed products (Fig. 6f). As indi- soned that the greatest impact would be mediated by those cated by the experiment with unfractionated exRNA, fragmen- miRNAs that are most abundant in EVs/exRNA, while not or tation is a feature of exRNA rather than an artifact of the minimally expressed in recipient cells, such that their transfer ﬁltration procedure (Supplementary Fig. 13). Of note, qRT-PCRs would signiﬁcantly alter their levels in recipient cells and thus quantiﬁcation of the selected transcripts correlated well with the affect the regulation of mRNA targets. To identify such miRNAs, RNAseq results (Fig. 6g), supporting the accuracy of our RNAseq we compared relative levels of all miRNAs in exRNA fractions analysis. from GSCs with those in the major types of human and mouse brain cells, including astrocytes, mature neurons, microglia, and MVs most closely reﬂect cellular RNA composition. Cancer- brain-derived endothelial cells. Comparison of three GSC extra- derived exRNA may serve as clinical biomarkers for disease cellular fractions with primary astrocytes is shown in Fig. 8a, and diagnostics, prognostics and monitoring. An extracellular fraction similar analysis for other cell types is presented in Supplementary that closely mirrors the deranged cellular transcriptome would be Fig. 18. Summary of this analysis, performed on three extra- the most valuable for such applications. To date, mostly unfrac- cellular fractions and four types of recipient cells, revealed a list of tionated, precipitated or 100,000 g-pelleted EVs have been the GBM-derived miRNAs that might potentially have the explored as a source for potential biomarkers. To examine what strongest impact on the normal cells of brain microenvironment type of extracellular complexes (MVs, exosomes, or RNPs) might (Fig. 8b). In agreement with these data, most of the listed serve as the closest proxy of the GSC transcriptome, we per- miRNAs were also found to be elevated in GBMs relative to the formed correlation and clustering analysis of their RNA com- control brain tissues in The Cancer Genome Atlas (TCGA) data position. Overall, clustering analysis based on small RNA libraries set (Fig. 8b). To predict the downstream effects of the 10 most demonstrated the highest similarity between the cellular tran- impactful miRNAs on the recipient cells, we analyzed their direct scriptome and MV content, and to a lesser extent to exosome mRNA targets previously validated by at least three supporting content, while the RNP fraction had a highly distinct RNA CLIP-Seq data sets in the starBase database . In total, 2267 composition (shown for GBM4 in Fig. 7a). This was further mRNA species interact with at least one of the impactful miR- conﬁrmed by principal component analysis (PCA) of all four NAs. Based on the ingenuity pathway analysis (IPA), these targets GSC cultures (Fig. 7b). Similar analyses of mRNA and miRNA are signiﬁcantly enriched in many canonical cancer-related classes, and the most abundant transcripts within them, further pathways and bioterms, including the molecular mechanism of supported this conclusion (Fig. 7c–f and Supplementary Figs. 14– cancer, ERK/MAPK signaling, PI3K/AKT signaling, and NGF 17). Consistent with this idea, there were fewer miRNA species, signaling (Fig. 8c). and overall fewer different RNA species signiﬁcantly enriched in MVs than in exosomes and RNPs (Supplementary Data 2). The Less than one copy per EV on average for most RNA species.A observation that MVs provide a more accurate peripheral read- substantial amount of transferred exRNA might be required to out of the source cell content is in line with recent extracellular exert a functional effect in a recipient cell; however, the quanti- proteomic analysis . Therefore, MVs which include large vesicles tative data for the levels of speciﬁc RNA classes and individual (200–800 nm) appear to be a good source for RNA biomarker transcripts in exRNAs is very limited thus far. To address this discovery, and have the potential to outperform the more studied issue, we performed a stoichiometric analysis of the smaller exosomes. EV-associated RNA. On average, GSC EVs (MVs and exosomes collectively) provided ~8.9 ng total RNA per ml of conditioned Analysis of exRNA complexes in cerebrospinal ﬂuid.To media. Considering the concentration 2 × 10 EVs per ml, mea- investigate the differential potential of exRNA fractions for bio- sured by NTA, one EV contained ~4.45 ag total RNA, or ~0.445 marker discovery, we tested our sequential ﬁltration method on ag non-rRNA, which corresponded to ~836 ribonucleotides. cerebrospinal ﬂuid (CSF) samples obtained from four GBM Similar analysis was performed for individual RNA classes and patients. As shown in Fig. 7g, sufﬁcient amounts of total RNA for species. As shown in Table 1, rRNA and snRNA are qRT-PCR analysis were isolated from each CSF fraction (MVs, present in more than one copy per EV on average. RNA classes exosomes, and RNPs), and distinct proﬁles observed for different represented by approximately one copy per EV include Y RNA RNA classes. Although we could not examine the resemblance of and Y RNA fragments, tRNA fragments, lncRNA, and miRNA. CSF exRNA proﬁles to those of parental tumor and neural cells One copy of mRNA or mRNA fragment can be found in ~10 EVs. due to unavailability of tumor material from the corresponding Of note, quantiﬁcation of individual RNA species revealed that patients, the data indicated that, indeed, MVs and exosomes only several speciﬁc molecules are present at the level of one copy should be analyzed separately in biomarker discovery studies. per EV (e.g., RNY1, Y5 fragments, GluCTC tRNA fragment, U1 Similarly to the results obtained on cultured GSC-derived exRNA, and U2 snRNA fragments). The most abundant miRNA species the CSF mRNAs were preferentially associated with MVs and (e.g., let-7b, miR-21) were present at the level of one copy per 10 miRNAs with exosomes, suggesting that MV enrichment is EVs approximately, consistent with the previous report . The warranted for mRNA biomarkers, while exosomes represent a most abundant individual mRNA species were present at one superior source for miRNA biomarkers. Of note, several mRNAs copy per 1000 EVs approximately (e.g., TMSB10), and the levels frequently mutated in GBM, including PTEN and COL1A2, were of mRNAs most commonly mutated in GBM (e.g., EGFR, IDH1, 10 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE TP53, PTEN, and COL1A2) were not higher than one copy per required to affect signaling or alter the phenotype of the recipient 100,000 EVs approximately. Based on these data, continuous cells via their exRNA content. barrage, massive and/or highly selective uptake of EVs might be To examine the functionality of transferred GSC-derived miRNAs, we established 3D transwell co-cultures of GBM8 neurospheres with primary mouse astrocytes, a system more physiological than the commonly used exposure of recipient Table 1 Quantiﬁcation of selected RNA classes and cultures to super-concentrated EVs isolated from donor cell individual species in copy number per EV conditioned medium. In line with the low copy number of individual miRNAs per EV, we were unable to detect signiﬁcant Copy level RNA class or individual species elevation of either miR-21 or miR-10b (top GBM-promoting More than 1 copy per EV RNA class rRNA fragment miRNAs) in astrocytes co-cultured with GBM8. However, Repeat fragment miR-21 became readily detectable in primary astrocytes estab- snRNA fragment lished from miR-21 knockout mice upon their co-culture with One copy per 1 EV RNA class tRNA fragment GBM8 (Fig. 8d). Furthermore, this latter miR-21 transfer led to approximately functional effects in these cells, i.e., repression of its previously Intron fragment validated mRNA targets (Fig. 8e) . These data support inter- Y RNA fragment SRP RNA fragment cellular transfer of miRNA via exRNA and suggest that, despite miRNA the apparently low levels in EVs, miRNAs may exert regulatory Exon fragment functions in recipient cells, albeit with speciﬁc conditions and lncRNA highly sensitive methodologies required for detection. Y RNA snoRNA fragment Individual RNA U2 snRNA fragment Discussion species exRNA studies have expanded rapidly and multidirectionally in U1 snRNA fragment this decade. Revealing the composition of exRNA complexes GluCTC tRNA fragment released by deﬁned cell types remains one of the most funda- RNY1 mental milestones toward understanding the role of exRNA in RNY5 fragment intercellular communication, as well as discovery of RNA bio- One copy per 10 EVs RNA class mRNA markers for disease. This study provides the ﬁrst minimally approximately biased quantitative analysis of the exRNA released by tumor- vRNA derived cells from GBM patients. Despite the high heterogeneity vRNA fragment of GSC cultures established from different tumors, and even Individual RNA RNY4 fragment higher heterogeneity of the exRNA they release, extracellular species complexes share key characteristics. The RNA proﬁles of MVs, RNY4 RNY1 fragment exosomes, and RNPs are highly distinct. They all display selec- let-7b-5p tivity in their RNA loading compared with the cells, with the MVs GlyCCC tRNA being the most like the cells and the RNPs the least. Small and fragment fragmented RNAs account for the majority of exRNA in all three miR-21-5p types of complexes. Between 64 and 93% of all exRNA consists of vRNA1-2 fragmented rRNA (Fig. 3a), although there is little-to-no intact vRNA1-2 fragment rRNA (Supplementary Fig. 3). miRNA, the most studied class of One copy per 100 EVs Individual RNA U3 snoRNA fragment exRNA, constitutes <10% of non-rRNA exRNA (Fig. 4a). Con- approximately species sistent with some previous publications , and in contrast to miR-1246 20, 37–41 others , we found that miRNA species are relatively enri- miR-10b-5p RNY5 ched in exosomes, but not in MVs or RNPs. Of note, the isolation vRNA1-1 fragment approaches, the library preparation strategies, and the normal- vRNA1-3 fragment ization methods utilized in these different studies were diverse, vRNA1-3 making the results not directly comparable. Other non-coding One copy per 1000 EVs Individual RNA RNY3 fragment RNA species are more abundant, and some of them are enriched approximately species in EVs or RNPs. Notable among them are precisely processed TMSB10 mRNA (top tRNA and Y RNA fragments, associated with both EVs and 1 mRNA) extravesicular RNPs, and supported by observations of other miR-93-5p 20, 37 42 cultured cells and body ﬂuids . One copy per 10,000 EVs Individual RNA ACTB mRNA approximately species The most common extracellular 30–32 nt-long 5′-tRNA frag- GAPDH mRNA ments (5′-tRFs), also called 5′-tRNA halves or tiRNAs, are evo- miR-132-3p lutionarily conserved molecules produced by angiogenin One copy per 100,000 EVs Individual RNA COL1A2 mRNA 44 (ANG), a member of ribonuclease A family . This multi- approximately species functional ribonuclease regulates angiogenesis, cell proliferation RNY3 and viability of cancer cells, as well as neuronal survival and stress miR-34a-5p response . Speciﬁc5′-tRFs are known to perform crucial func- IDH1 mRNA tions, often associated with regulation of gene expression in stress EGFR mRNA response. They repress protein translation by displacing eukar- 1 copy per 1,000,000 EVs Individual RNA TP53 mRNA yotic translation initiation factors eIF4E and eIF4G ; and mod- approximately species PTEN mRNA ulate stress response by inducing formation of stress granules— cytoplasmic foci where untranslated mRNAs are transiently The transcripts were deﬁned as fragments if present and quantiﬁed in small RNA libraries stored . Importantly, speciﬁc5′-tRFs (e.g., 5′Ala, NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 11 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x 5′His, and 5′Cys) selectively regulate translation of subsets of nuclear proteins PCNA and Ro60 appeared enriched in RNPs but mRNAs, both capped and uncapped, and therefore reprogram not in EVs. While the co-localization of Ro60 with its binding Y protein synthesis . They may also function in a miRNA-like RNA partners in RNPs may point to the functionality of this 46 47, 48 manner . ANG is upregulated in GBM and is one of the extracellular complex, the role of extracellular PCNA, a protein proteins most highly secreted by glioma cells . Despite its high involved in DNA replication, remains to be elucidated. Although abundance, functions of ANG, including its extracellular activity the possibility of direct non-vesicular, RNP-mediated RNA and its exRNA products remain to be investigated. Remarkable uptake and function in recipient cells is currently unexplored, it is 61, 62 enrichment of both ANG and 5′-tRFs observed in GSC-derived supported by the utility of RNPs for RNA and drug delivery . exosomes (Figs. 2b and 6f) suggests that tRNA cleavage may Regardless of the potential biological function of RNPs, their occur in exosomes, outside of the cells. associated RNAs expand the repertoire of potential biomarkers Another highly abundant but poorly studied classes of that should be further explored in body ﬂuids, in parallel to EV non-coding exRNAs are Y RNAs and their speciﬁc5′ fragments. transcripts. The majority of information about Y RNA has come from studies The key outcome of our work, overall, is an expansion of the of bacteria and invertebrates. In vertebrates, Y RNAs are repertoire of exRNAs released by GSCs in different vehicles, with expressed in all tissues and cells and have been proposed to functional and biomarker potential, far beyond the class of 50, 51 participate in many important cellular functions . Their lower miRNA. This conclusion challenges the commonly assumed stem domain, which recruits chaperone Ro60 and exoribonu- predominant role of miRNA in exRNA-mediated intercellular clease PNPase, may play a role in RNA quality control and communication. It further points to the need for in-depth degradation of misfolded RNAs . The upper stem domain may investigation of other classes of exRNAs and their impact on the participate in initiation of chromosomal DNA replication . With physiology of recipient cells and use as biomarkers. The future biogenesis and activity independent of Dicer and Ago2 , Y RNA should bring the development of novel experimental techniques fragments do not appear to silence gene expression in a miRNA- and computational resources for integrating complex expression like manner . Recent studies reported that Y RNA fragments data sets into comprehensive biologic networks and biomarker may be involved in histone mRNA processing and cell discovery. damage . Interestingly, Y5 fragment, the most abundant in exRNA, is proposed to specialize in surveillance of ribosomal Methods RNA . Studying the functions of these precisely processed, GBM stem cell cultures. Human low-passage (below 20) GBM cells (kind gift from Dr. Hiroaki Wakamoto, MGH) were cultured as neurospheres in Neurobasal highly enriched extracellular transcripts represents an exciting medium (Gibco) supplemented with 3 mM GlutaMAX (Gibco), 1× B-27 supple- new avenue in RNA biology. ment (Gibco), 0.5× N-2 (Gibco), 20 ng/ml EGF (R&D systems, MN), 20 ng/ml FGF In addition to non-coding RNA, we detected low levels of (PEPROTECH, NJ) and 0.5% Antibiotic-Antimycotic Solution (Corning), and mRNA reads with UTRs relatively enriched compared to the passaged by NeuroCult Chemical Dissociation Kit (Mouse) (Stemcell Technologies, Canada) following the manual. Approximately 5 × 10 dissociated cells were seeded coding regions in exRNA. The mechanism underlying this per 10 cm dish (Corning) in 10 ml fresh media, and 1/3 volume of fresh medium enrichment and its biological impact has yet to be investigated. was added every 3 days. Mature neurospheres, typically formed in 7–10 days, were 58, 59 Methylation and other RNA modiﬁcations of UTRs might dissociated and replated. All cells were tested for mycoplasma. Human cells were cause differential sorting or stability, leading to enrichment in used in accordance with the policies of institutional review boards at Brigham and Women’s Hospital. exRNAs. UTRs and their fragments might function as molecular sponges for various regulatory molecules, including miRNA, translation factors (e.g., eIF4F and ribosomal complexes), other Primary cultures of normal brain cells. Brain cortices of E18 and P1 C57BL/6 mice were dissected for primary cultures of neurons and glial cells, respectively. RNA-binding proteins, and thereby exert functions in recipient The tissues were dissociated with 0.25% Trypsin (Gibco) and 0.1 mg/ml DNaseI cells. Altogether, our data indicate the strong preference of pro- (Roche) for 15 min at 37 °C. The cells were plated in poly-D-lysine-coated T25 cessed fragments for multiple classes of RNA, protein-coding and 2 ﬂasks or 24-well plates at ~80,000 cells per cm , in the seeding medium consisting non-coding, in exRNA (Fig. 6). The co-packaging of of DMEM-F12 (Corning), 10% FBS (Gibco), and 1% Antibiotic-Antimycotic Solution (Corning). For neuronal cultures, the media was exchanged to Neurobasal processed RNA with various RNA-binding proteins, including (Gibco), 2% B-27 (Gibco), 1% Antibiotic-Antimycotic Solution (Corning) and 0.5 RNases (e.g., ANG) and effecter complexes (e.g., Ago2), and the mM Glutamax (Gibco) 1 day after plating. Mature neurons at 21 days in culture signiﬁcant depletion of full-length RNA in exosomes, suggests have been utilized for the RNAseq. For glial cultures, the ﬂasks were shaken (200 that they may function as an exRNA processing machinery with rpm at 37 °C) three times overnight to remove microglia, and astrocytes trypsinized and further cultured in 24-well plates. For microglia cultures, the media was specialized autonomous functions. supplemented with the recombinant M-CSF mouse protein (10 ng/ml; Gibco). Although several studies have explored the RNA bound to Floating microglial cells were collected from the conditioned media by gentle spin speciﬁc secreted or circulating proteins, such as high-density (300×g, 10 min), and re-plated in 24-well poly-D-lysine-coated plates at ~100,000 3 60 lipoprotein (HDL) and Ago2 , overall little attention has been cells per cm . For human primary neuroglial cultures, fetal cortical tissues paid thus far to the non-vesicular exRNA complexes that account (gestational age 18 weeks) were provided by Advanced Bioscience Resources, Inc. (Alameda, CA), dissociated with papain (12 U/ml; Worthington), seeded with for the signiﬁcant proportion of exRNA (Fig. 2c). We present data neuronal media plus 2% FBS (Gibco), and cultured as mouse neurons . Neuronal indicating that extracellular non-vesicular RNPs exhibit a RNA cultures at 30 days in vitro have been utilized for the RNAseq. Human (HBEC) and signature readily distinguishable from EVs. Notably, ncRNA mouse (MBEC) primary brain microvascular endothelial cells were purchased from accounts for nearly entire RNA population in RNPs, with a large Cell Biologics, IL (Catalog# H-6023; Lot# 021514F14 and Catalog# C57-6023; Lot# 070613T2MP, respectively) and cultured accordingly to the manufacturer. All prevalence of tRNA, Y RNA and their products, and depletion of animal experiments have been approved by the Harvard Medical Area Standing miRNA species (Figs. 4 and 6), consistent with the analysis of Committee on Animals. breast cancer extracellular RNPs . Although extracellular RNPs might be highly heterogeneous, this collective data suggest the key Fractionation and RNA isolation. Approximately 100 ml conditioned medium pathways involved in the biogenesis. Of note, we found that was used as input for exRNA isolation. Conditioned media was centrifuged at massive levels of albumin, an abundant component in B-27 300×g, 4 °C for 10 min, following by the additional centrifugation at 2000×g,4°C for 15 min, to remove cells and cell debris. To monitor EVs, the samples were supplement, hamper the protein characterization of extracellular diluted in DPBS and examined using the Nanoparticle Tracking Analysis system RNPs, and markers of RNPs have yet to be deﬁned. Beneﬁting (NanoSight LM10; Malvern Instruments, UK), and EV concentrations were from an efﬁcient (>95%) albumin-depletion protocol, we were quantiﬁed within the optimal linear range (2–10 × 10 particles per 1 ml). able to compare protein content in MVs, exosomes, and For RNA preparation, 5 μl of SUPERase In RNase Inhibitor (Ambion) was non-vesicular RNPs using immunoblots. Interestingly, two added to the supernatants per 10 ml media. The media was then ﬁltered 12 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE sequentially through the 2 μm ﬁlter (GE Healthcare, UK), 0.8 μm ﬁlter (EMD performed by Cuffdiff with parameter -u/--multi-read-correct and normalized Millipore, MA), and 0.22 μm ﬁlter (EMD Millipore), with no/minimal pressure based on the ERCC spike-in controls using the cyclic loess robust local regression. applied. The ﬁltrate was split to 15 ml per sample and further ﬁltered through the Clean reads produced from the small RNA libraries were ﬁrst aligned to carrier 0.02 μm ﬁlter (GE Healthcare) with up to 75 psi pressure applied. To facilitate the RNA (Enterobacteria phage MS2) of miRCURY Spike-in kit with bowtie 0.02 μm ﬁltration, a mechanical syringe pump was designed and manufactured (parameter: -v 2 -l 7 --all). The unmapped reads were aligned against human rRNA (Supplementary Fig. 2). Upon ﬁltration, each ﬁlter was washed with 1 ml DPBS sequences with bowtie (parameter: -v 2 -l 7 --all) to remove the remaining rRNA (Corning), and the corresponding fractions were lysed with 600 μl lysis solution of reads. The remaining reads were mapped to human miRNA precursors (miRBase the miRCURY RNA Isolation Kit—Cell & Plant (Exiqon, Denmark). The fractions V19) with bowtie (parameter: -v 1 --all), and the miRNA precursors reads were collected on 0.02 μm ﬁlters were lysed with 900 μl lysis solution. The last ﬂow- separated according to the mapping position. The mapped reads with more than through fractions of 0.02 μm ﬁlters were pooled together (up to 30 ml) and 10% mismatched nucleotides were excluded. The remaining reads were mapped to concentrated ~60 times using 3 kDa Amicon Ultra Centrifugal Filters (EMD spike-ins (UniSp2, UniSp4 and UniSp5; parameter: -v 1 --all), and other non- Millipore) at 4000×g, 4 °C, for 60 min. The concentrates were collected and lysed coding RNA classes including tRNAs (gtRNAdb), piRNAs (RNAdb), snoRNA with six volumes of the same lysis solution (Exiqon). Total RNA was then isolated (snoRNA-LBME-db), scRNAs (Genbank), and others (Rfam database) (parameter: from all fractions as recommended by miRCURY protocol, with -v 2 --all). Finally, the remaining reads were mapped to the hg19 human genome, on-column DNase treatment (Qiagen, Germany). The corresponding 1.2 ml of the and the reads mapped to exons, introns, intergenic regions, and repeats (parameter: source neurospheres were span down at 300×g, 4 °C for 5 min, and total cellular -v 0 --all) identiﬁed. The SAMtools was used to calculate the reads depth for each RNA was isolated from them and analyzed in parallel. The same protocol was base position and R package barplot was used to draw the corresponding bar plot. carried out for RNA isolation from fresh media, with 500 ml media input. For RNA isolation from primary cells cultured in 24-well plates, the cells were lysed with 350 μl lysis solution per well. The concentrations of cellular and extracellular RNA were Data analysis. Based on the known amounts of spike-in RNAs, the read count for determined using the NanoDrop 2000 Spectrophotometer and Quant-iT each RNA species was ﬁrst normalized to spike-ins’ reads, to quantify the absolute RiboGreen RNA Assay Kit (Thermo Fisher Scientiﬁc), respectively. The RNA amounts in fmol per μg of total RNA. Next, the obtained values were corrected to quality was examined using Agilent 2100 Bioanalyzer (Agilent, CA) and the RNA the corresponding fresh media as the blank control, using the equation shown in Integrity Number (RIN) estimated. Supplementary Fig. 6. For the analysis of class composition, the total abundance of corrected non-rRNA was used for normalization between the samples. To compare heterogeneity of the samples, the sum of squared errors (χ value) of species Transmission electron microscopy. The material collected on the ﬁlters was composition was calculated using MS Excel Macro (available in Supplementary resuspended using DPBS (Corning), and further pelleted by 100,000×g UC for 80 Data 3). To estimate the inequality of abundance among all RNA species in one min at 4 °C. The material diluted in DPBS was added to a glow-discharged carbon- class, the evenness factor, Gini coefﬁcient, and traditional pre-set evaluations were coated grid. The grids were washed with distilled water, stained with 0.75% uranyl calculated using a MS Excel Macro (available in Supplementary Data 4). The formate, examined using Tecnai G Spirit BioTWIN microscopy (FEI, OR), and hierarchical clustering analysis was performed using the MultiExperiment Viewer images recorded by the AMT 2k CCD camera (Advanced Microscopy Techniques, (Dana-Farber Cancer Institute, MA). The principle component analysis (PCA) was MA) at the Harvard Medical School EM Facility. performed and visualized with R package rgl. miRNA targets were determined based on starBase v2.0, with at least three supporting CLIP-Seq experiments . RNA sequencing. Two sets of spike-in RNAs were added to the samples prior to Pathway analysis of target mRNAs was performed using the Ingenuity Pathway library preparation: ERCC RNA Spike-In Control Mixes (Ambion; 0.02 μl per 1 μg Analysis (IPA; Qiagen). Venn diagrams were plotted using Venny 2.1. total RNA), and miRCURY Spike-in kit, part 1, with UniSp2’s ﬁnal concentrations of 1.25 and 5.0 fmol per 1 μg of total RNA for cellular and extracellular RNA, respectively. Total RNA, either 40–200 ng of exRNA, or 2 μg of cellular RNA, was Long RNA reverse transcription PCR. Maxima Reverse Transcriptase (100U; rRNA-depleted using the Ribo-Zero rRNA Removal Kits (Illumina, CA). One Thermo Fisher Scientiﬁc) was used to reverse transcribe 20 ng RNA with both oligo quarter of the rRNA-depleted RNA was fragmented to 100–500 nt using the 5× (dT) and random hexamers in a 10 μl reaction system. Next, 0.5 μl cDNA was used First-Strand Buffer (Clontech, CA), and utilized for the long RNA library con- in 10 μl PCR reactions based on Phire Hot Start II PCR Master Mix (Thermo struction by SMARTer Stranded RNA-Seq Kit (Clontech). The remaining 75% of Fisher Scientiﬁc) and primers (0.5 μM each), and ampliﬁed using a touchdown the rRNA-depleted RNA was treated sequentially with Tobacco Acid Pyropho- program with either 15 or 70 s extension step for short or long ampliﬁcation, sphatase (TAP; Illumina) and T4 Polynucleotide Kinase (T4PNK; New England respectively. The PCR products were examined on 0.8–1.2% agarose gels (Thermo Biolabs, MA) to create more uniform 5′- and 3′-ends for various classes of tran- Fisher Scientiﬁc). Primer sequences are provided in Supplementary Table 3. scripts. The RNA was then used as input for the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs), with size selection of 15–65 nt inserts for small RNA libraries. The quality of libraries was examined using the Quantitative reverse transcription PCR. Generally, for small RNA qRT-PCR, 10 Agilent DNA 1000 kit at the Agilent 2100 Bioanalyzer instrument, and cDNA ng of total RNA was used in 10 μl reverse transcription reaction with Universal quantiﬁed by qRT-PCR. The libraries were sequenced on HiSeq 2000 (Illumina) cDNA Synthesis kit II (Exiqon). The cDNA was diluted 80 times, and 4 μl was used with single read 50 cycles by Beijing Genomics Institute (BGI, China). in 10 μl qPCR reactions using the ExiLENT SYBR Green master mix and custom- designed LNA primers (Exiqon). For mRNA qRT-PCR, 10 ng of total RNA was used in 10 μl reverse transcription reaction with PrimeScript RT Master Mix Reads annotation. Sequencing reads were treated using the BGI pipeline that included multiple ﬁltering steps, as follows: (1) removing reads with adapters; (2) (Takara, Japan). One microliter cDNA was used in 10 μl qPCR reactions using the ExiLENT SYBR Green master mix and synthesized primers (Supplementary removing reads with >10% of unknown bases; and (3) removing low-quality reads Table 4; Eton Bioscience, CA). The qPCR reactions were run on a ViiA 7 (sequencing quality <10). After ﬁltering, the remaining clean reads were subjected instrument (Thermo Fisher Scientiﬁc) in duplicates. The speciﬁcity of qPCR to the bioinformatics analysis. Generally, 20 and 10 M clean reads were generated products was veriﬁed by the presence of a single peak at the melting curves. per long RNA library and small RNA library, respectively. The clean reads pro- duced from the long RNA libraries were ﬁrst mapped to the Homo sapiens rRNA database using the SOAPaligner/SOAP2 short read alignment software, to remove the remaining rRNA reads. The non-rRNA reads were used to perform the tran- Immunoblotting. For protein analysis, cellular and extracellular fractions were scriptome assembling and quantiﬁcation. First, non-rRNA reads were mapped to lysed using the modiﬁed RIPA buffer containing 2% SDS, 1% sodium deoxycholate human reference genome hg19 using an improved version of TopHat2, which and 3 M urea. The RNP fractions were BSA-depleted using the Aurum Afﬁ-Gel aligns reads across splice junction without relying on gene annotation. Default -g/-- Blue Mini Columns (Bio-Rad, CA), and concentrated using Pierce SDS-PAGE max-multihits was used to allow up to 20 multimapping. Next, the reads mapped Sample Prep Kit (Pierce, MA). Protein concentrations were quantiﬁed using the to genome were assembled using Cufﬂinks. Reference Annotation Based Transcript Micro BCA Protein Assay Kit (Pierce). Equal amounts of the total protein (50 μg) (RABT) assembly was performed with the reference gene annotation to compen- were loaded per lane in Novex WedgeWell 14% Tris-Glycine Mini Gel (Thermo sate incompletely assembled transcripts caused by read coverage gaps in the regions Fisher Scientiﬁc). Proteins were transferred to 0.45 μm PVDF membrane (Thermo of reference gene. The set of transfrags generated was then compared with the Fisher Scientiﬁc). After blocking with 5% (wt/vol) fat-free milk in Tris-buffered reference transcripts to remove transfrags that were approximately equivalent to saline with 0.075% Tween-20 (TBST), membranes were incubated with 1:1000 the whole or a portion of a reference transcript. After the assembling, the whole diluted primary antibodies (Flotillin-1 #18634S, CD9 #13174S, Integrin β1 #9699S, parsimonious set of transcripts was obtained. These transcripts were blasted with HSP90 #4874S, La #5034S, NPM #3542S, Ago2 #2897S, Alix #2171S, and PCNA the NONCODE database using the ﬁlter set (identity >0.9 and coverage >0.8) to #13110S from Cell Signaling Technology, MA; Ro60 #AV40534 from Sigma- identify known long non-coding RNA. Then, the rest of assembled transcripts were Aldrich; ANG #sc-74528 from Santa Cruz Biotechnology, TX) overnight at 4 °C. aligned to the reference annotation utilizing Cuffcompare. Thereafter, Cuffmerge The membranes were washed and incubated with horseradish was utilized to merge several assemblies from different samples together, which peroxidase–conjugated secondary antibodies (#7074S and #7076S from Cell Sig- automatically ﬁltered out a number of transfrags that probably were artifacts and naling Technology, 1:2000 dilution) for 1 h at room temperature. The blots were produced a single annotation ﬁle for downstream gene expression analysis. Two developed by the Amersham ECL Reagent (GE Healthcare) and, if required, mismatches were allowed for annotation generally, and only one mismatch was stripped using One Minute Western Blot Stripping Buffer (GM Biosciences, CT). allowed for the ERCC spike-ins. mRNA and lncRNA expression analyses were Uncropped scans of blots are shown in Supplementary Fig. 19. NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 13 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x Transwell cocultures of GSC and astrocytes. Mouse cortical astrocytes estab- 14. Teplyuk, N. M. et al. Therapeutic potential of targeting microRNA-10b in lished from WT or miR-21 KO P1 pups, passage 1 or 2, were cultured in established intracranial glioblastoma: ﬁrst steps toward the clinic. EMBO Mol. 24-well plates. 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We thank Robert Kitchen, Sai Lakshmi Subramanian, Alain Charest, Steven associate with AGO proteins to recognize speciﬁc RNA targets. BMC Biol. 12, Gould, Thorsten Mempel, and the RNAseq working group of the Extracellular RNA 78 (2014). Communication Consortium for valuable insights on the data analysis. We thank Maria 47. Eberle, K. et al. The expression of angiogenin in tissue samples of different Ericsson for TEM, Hongjun Wang for designing and manufacturing syringe pumps, brain tumours and cultured glioma cells. Anticancer. Res. 20, 1679–1684 (2000). Pavel Ivanov for sharing ANG antibody, Oleg Butovsky for sharing miR-21 knockout 48. Xia, W. et al. Angiogenin promotes U87MG cell proliferation by activating NF- mice, and Erik J. Uhlmann for reviewing the manuscript. We thank members of Kri- kappaB signaling pathway and downregulating its binding partner FHL3. PLoS chevsky and Breakeﬁeld laboratories for helpful discussions. ONE 10, e0116983 (2015). 49. Skog, J. et al. 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Genes Dev. 29, 1998–2003 (2015). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in 56. Chakrabortty, S. K., Prakash, A., Nechooshtan, G., Hearn, S. & Gingeras, T. R. published maps and institutional afﬁliations. Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA. RNA 21, 1966–1979 (2015). 57. Hogg, J. R. & Collins, K. Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control. Genes Dev. 21, 3067–3072 (2007). Open Access This article is licensed under a Creative Commons 58. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals Attribution 4.0 International License, which permits use, sharing, enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012). adaptation, distribution and reproduction in any medium or format, as long as you give 59. Ke, S. et al. A majority of m6A residues are in the last exons, allowing the appropriate credit to the original author(s) and the source, provide a link to the Creative potential for 3′ UTR regulation. Genes Dev. 29, 2037–2053 (2015). Commons license, and indicate if changes were made. The images or other third party 60. Arroyo, J. D. et al. Argonaute2 complexes carry a population of circulating material in this article are included in the article’s Creative Commons license, unless microRNAs independent of vesicles in human plasma. Proc. Natl Acad. Sci. indicated otherwise in a credit line to the material. If material is not included in the USA 108, 5003–5008 (2011). article’s Creative Commons license and your intended use is not permitted by statutory 61. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. 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Cellular and Molecular Life Sciences REVIEW Membrane vesicles, current state-of-the-art: emerging role of extracellular vesicles
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Publisher: Springer Journals
Copyright: Copyright © 2017 by The Author(s)
Subject: Science, Humanities and Social Sciences, multidisciplinary; Science, Humanities and Social Sciences, multidisciplinary; Science, multidisciplinary
eISSN: 2041-1723
DOI: 10.1038/s41467-017-01196-x
pmid: 29074968
Publisher site: See Article on Publisher Site

Abstract

ARTICLE DOI: 10.1038/s41467-017-01196-x OPEN Coding and noncoding landscape of extracellular RNA released by human glioma stem cells 1 2 3 4 1 1 Zhiyun Wei , Arsen O. Batagov , Sergio Schinelli , Jintu Wang , Yang Wang , Rachid El Fatimy , 1 5 6 7,8 7 5 Rosalia Rabinovsky , Leonora Balaj , Clark C. Chen , Fred Hochberg , Bob Carter , Xandra O. Breakeﬁeld & Anna M. Krichevsky Tumor-released RNA may mediate intercellular communication and serve as biomarkers. Here we develop a protocol enabling quantitative, minimally biased analysis of extracellular RNAs (exRNAs) associated with microvesicles, exosomes (collectively called EVs), and ribonucleoproteins (RNPs). The exRNA complexes isolated from patient-derived glioma stem-like cultures exhibit distinct compositions, with microvesicles most closely reﬂecting cellular transcriptome. exRNA is enriched in small ncRNAs, such as miRNAs in exosomes, and precisely processed tRNA and Y RNA fragments in EVs and exRNPs. EV-enclosed mRNAs are mostly fragmented, and UTRs enriched; nevertheless, some full-length mRNAs are present. Overall, there is less than one copy of non-rRNA per EV. Our results suggest that massive EV/exRNA uptake would be required to ensure functional impact of transferred RNA on brain recipient cells and predict the most impactful miRNAs in such conditions. This study also provides a catalog of diverse exRNAs useful for biomarker discovery and validates its feasibility on cerebrospinal ﬂuid. Department of Neurology, Brigham and Women’s Hospital and Harvard Medical School, HMS Initiative for RNA Medicine, Boston, MA 02115, USA. 2 3 Vishuo Biomedical, #3-33 Teletech Park, 20 Science Park Road, Singapore 117674, Singapore. Department of Drug Sciences, University of Pavia, Pavia 4 5 27100, Italy. Beijing Genomics Institute, Shenzhen 518083, China. Department of Neurology and Radiology, Massachusetts General Hospital and Program in Neuroscience, Harvard Medical School, Charlestown, MA 02129, USA. Neurosurgery Department, University of Minnesota, Minneapolis, MN 55455, 7 8 USA. Department of Neurosurgery, University of California, La Jolla, San Diego, CA 92093, USA. Scintillon Institute, San Diego, CA 92121, USA. Correspondence and requests for materials should be addressed to A.M.K. (email: [email protected]) NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 1 | | | 1234567890 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ntercellular communication within complex biological sys- investigation of cancer-derived exRNA. As proliferating and tems, such as cancer and its host microenvironment, via invading GBM cells migrate through brain parenchyma, they I“horizontal” RNA transfer, is an expanding area of research . interact with the changing landscape of extra-tumoral stimuli and Extracellular RNAs (exRNAs) are packaged into various extra- actively modulate this landscape . Communication between cellular complexes, including microvesicles (MVs), exosomes, and tumor cells and diverse normal cells in the brain is nevertheless 2, 3 non-vesicular ribonucleoprotein complexes (RNPs) . MVs and one of the least investigated aspects of glioma biology. We exosomes, broadly called extracellular vesicles (EVs), are released employed low-passage patient-derived tumorigenic GBM cell and taken up by various cells, thereby transferring their content. cultures that represent the most therapy-resistant stem-like cell This process likely plays a role in cancer development and population (GSC), and are considered the core cell type within manipulation of its microenvironment . However, methodologies the tumor. Analysis of GSC cellular and extracellular RNA, along are only beginning to emerge for characterizing the exRNA with the transcriptome of primary human and mouse cells of the landscape and monitoring levels of individual coding and reg- brain microenvironment (neurons, astrocytes, endothelial cells, ulatory exRNAs. exRNA mostly consists of small RNA species and microglia) enables us to predict the most impactful miRNAs (<200 nt); and the majority of reports to date focus on and expand the repertoire of potentially transferred exRNAs far 5, 6 miRNA . As a critical step toward understanding the biological beyond the classes of miRNAs and mRNAs. We also demonstrate impact of exRNA release and transfer, we investigated the com- that MVs, large vesicles of 0.2–0.8 μm, most closely mirror the plete spectrum of cancer-derived exRNAs, and the enrichment of cellular transcriptome and thus present a highly promising but speciﬁc RNA classes and individual species. By creating cDNA yet poorly explored source of liquid biopsy biomarkers. libraries of both small and long exRNA, and reducing the ligation bias favoring miRNAs, we identiﬁed a diverse and highly distinct composition of exRNA in MVs, exosomes, and RNP complexes. Results Furthermore, semi-absolute quantiﬁcation of RNAseq, performed Sequential ﬁltration-based exRNA isolation. To characterize using RNA spike-in molecules, allowed us to monitor the levels of exRNA released by patient-derived GBM cells in various com- various RNA classes and species in these extracellular complexes. plexes, we assessed several technical approaches. EV and exRNA This work focused on glioblastoma (GBM), the most common isolation protocols can be generally categorized into three major and aggressive brain tumor, as an important model for groups: based on ultracentrifugation (UC), precipitation using ab d 0.22 μm 0.02 μm RNA sequencing pipeline RNA isolation pipeline 40–2000 ng RNA Cell Conditioned media rRNA depletion NS 300 g 10 min ** 25 NS UC 8–25% 92–75% 2000 g 15 min Filtration 0 0 SMARTer for long RNA TAP and T4PNK Add RNase inhibitor (with fragmentation) treatment 2.0 μm filtration NEBNext for small RNA HiSeq2000 SR50 c Advantages of filtration based exRNA isolation (select 15–65 nt insert) Sequential filtration 0.8 μm filtration superior to: Benefits rRNA mapping HiSeq2000 SR50 MV Clearer separation 0.22 μm filtration >100 folds lower pressure on EV mRNA mapping rRNA mapping Higher yield Exosome 0.02 μm filtration No external chemical Less variation between batches miRNA mapping ncRNA mapping Require no capital equipment RNP 3 kDa concentration Applicable to big volume Abbreviations: tRNA, Y RNA, Rfam RNA isolation with UC: Ultracentrifugation PP: Polymer precipitation and other mappings DNase treatment DG: Density gradient UC GF: Gel filtration Fig. 1 Flowchart of the exRNA fractionation and sequencing. a The pipeline of the ﬁltration-based exRNA isolation. Following removal of cells and cellular debris by low speed centrifugation, the supernatants were ﬁltered through a sequence of reduced pore sizes (2.0, 0.8, 0.22, and 0.02 μm) to separate the extracellular fractions, and a ﬁnal concentrator with the cutoff of 3 kDa was applied to collect the remaining small particles. b The aliquots of conditioned media after 0.8 μm ﬁltration were used for MV and exosome isolation, either by ultracentrifugation (UC) or ﬁltration, and the RNA yield of these fractions compared. The number of remaining vesicles/particles was compared in UC supernatant and ﬁlter ﬂow-through. N = 4 aliquots of conditioned media. All bars represent mean ± SEM. c Comparison between the ﬁltration-based exRNA isolation and other common exRNA isolation methods. The stars mark superior characteristics of sequential ﬁltration over other methods. d The optimized pipeline for the broad coverage, minimally biased RNA-sequencing. RNA of 15–65 nt was selected for the small RNA libraries, to reduce the overwhelming levels of tRNAs. NS, not signiﬁcant; *p < 0.05; **p < 0.01; t-test 2 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | Isolated MV Isolated exosome After MV isolation After exosome isolation RNA concentration (ng/ml media) 0.8 μm filtrate 16,000 g 0.5 h Vesicle/particle number (*10 per ml) 100,000 g 2.5 h NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE ab Ladder (kDa) Cell MV Exosome RNP MV Exosome RNP 52- Flotillin-1 26- CD9 ANG (angiogenin) 10- Integrin β1 140 - 0.5 μm 0.1 μm 0.1 μm 95- HSP90 52- La antigen MGG75 GBM8 NPM GBM4 34- (nucleophosmin) 20/3 Ago2 95- 5 (argonaute 2) 140 - Alix 95- PCNA 34- (proliferating cell 26- nuclear antigen) 72- Ro60 (TROVE2) 52- Fig. 2 Quality control of the fraction separation. a Transmission electron microscopy of EVs and RNPs isolated using the sequential ﬁltration protocol. TEMs were replicated three times. b Protein markers, identiﬁed by the western blotting, veriﬁed the separation of extracellular fractions. Equal protein input of 50 μg per lane was used. Western blots were replicated twice. c RNA yields of extracellular fractions from different GSC cultures chemical polymers (PP), such as polyethylene glycol, and frac- different patients varied in the amount of exRNA released tionation, including density gradient UC and gel ﬁltration (Fig. 2c), ranging between 5.4 and 38.0 ng/ml accumulated in (DG&GF) . Since speciﬁc markers or physical parameters for the culture over 7 days. Considering that 3–10 μg of cellular RNA was various types of EVs and extracellular RNPs are still not clearly isolated from 1 ml of the corresponding GSC neurosphere deﬁned, UC remains the most commonly used approach to iso- cultures (~1.3 million cells), between 0.05 and 0.7% of cellular late the entire vesiculome . However, based on nanoparticle RNA is accumulated in the extracellular space in 7 days. Of note, tracking analysis (NTA; NanoSight) and ﬂuorescent dye-binding total exRNA yield varied ~7-fold among the GSC cultures, and quantiﬁcation (RiboGreen), the yield of EVs and exRNA isolated the proportion of exRNA associated with different extracellular by this technique is relatively low (20–40%) (Supplementary complexes also varied between the GSC types, suggesting the Fig. 1). Furthermore, this procedure yields a highly heterogeneous variations between cultures reﬂected intrinsic properties of 9, 10 mix of EVs and RNP/LNP (liponucleoprotein) particles .To different tumors. Analytical RNA proﬁles examined by the separate EVs and RNPs according to their physical size and Agilent 2100 Bioanalyzer indicated high-quality cellular RNA improve the yield of exRNA isolation, we developed a sequential (RIN > 9.5) with sharp rRNA peaks and no sign of degradation ﬁltration (SF) protocol (Fig. 1a). This protocol offers several (Supplementary Fig. 3). In contrast, exRNA exhibited mostly advantages over current methods, including low pressure on EVs, short RNA proﬁles (below 200 nt) with intact rRNA peaks better separation between EVs and RNPs, higher RNA yield, and detectable in large MVs, but not exosomes and RNPs. scalability (summarized in Fig. 1b, c and Supplementary Fig. 1). However, the extended hands-on time of the ﬁltration procedure (Supplementary Fig. 2) and the separation solely on the basis of Technical considerations for RNAseq. The protocols commonly size are limitations of this method. Also, retrieval of EVs from the utilized for small RNA library construction are based on adaptor ﬁltration membranes is inefﬁcient and could potentially alter their ligations to the 5′-phosphate and 3′-hydroxy ends of RNA, the structure; therefore, the utility of this protocol for functional EV modiﬁcations characteristic for miRNA, and thus favor analysis needs additional evaluation. miRNA . In order to characterize RNA content in a minimally Established glioma cell lines have very limited capacity to biased way, we utilized sequential treatments with tobacco acid reﬂect GBM biology . We utilized previously characterized low- pyrophosphatase (TAP) and T4 polynucleotide kinase (T4 PNK) passage GSC cultures derived from four primary human to create more uniform 5′ and 3′ ends for various types of 12, 13 heterogeneous GBM tumors for the exRNA proﬁling . These transcripts, leading to their more accurate representation in the 16, 17 cells were grown as neurospheres in serum-free medium, to cDNA libraries . The caveat is that this end-modifying pro- maintain their initial properties and transcriptional proﬁles and, cedure leads to an overwhelming abundance of rRNA reads in therefore, better reﬂect tumor biology . Transmission electron cellular and exRNA samples, and reduces the sequencing depth microscopy conﬁrmed the presence of EVs/particles in the for other RNA classes. Therefore, we included an rRNA depletion corresponding extracellular fractions isolated from GSC cultures step in the protocol that reduced rRNA reads remarkably (Fig. 2a). Distinct proﬁles of several protein markers exhibited by (Fig. 3a). cellular and extracellular fractions served to conﬁrm the purity of Unlike the established strategies for normalization of cellular fractions and the lack of cellular contamination in the MV and RNAseq data sets, which utilize total mapped reads as the other extracellular fractions (Fig. 2b). GSC cultures derived from normalization factor, there are no adequate standard for NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 3 | | | MV Exosome RNP RNA concentration (ng/ml media) RNP-enriched Exosome-enriched MV-enriched ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x a b 14 Without rRNA depletion With rRNA depletion Conditioned media Long RNA library Small RNA library Long RNA library Small RNA library Fresh media 9.7% 27.4% 8 MV MV MV MV long RNA small RNA long RNA small RNA library library library library 89.6% 85.7% 2 MV Exosome RNP 9.5% 36.7% Exosome Exosome Exosome Exosome long RNA small RNA small RNA c long RNA library library library library 92.5% 90.5% MV Exosome RNP 33.5% RNP RNP RNP RNP long RNA small RNA long RNA small RNA library library library library 92.8% 63.9% Before After rRNA reads Non-rRNA reads correction correction Fig. 3 rRNA depletion and media correction are warranted for the deep RNAseq analysis. a Comparison of the exRNA libraries prepared with and without rRNA depletion. The percent of rRNA reads is shown, indicating that the majority of exRNA reads in non-depleted libraries represent rRNA. Sequencing depth of other exRNA classes is substantially increased by the rRNA depletion. b Quantiﬁcation of total RNA in conditioned and fresh media indicates that the vast majority is cell derived (n = 4 GSC cultures). Nevertheless, quantiﬁcation of speciﬁc RNA species can be skewed by the media, as illustrated in c. The levels of miR-122 were assessed in exRNA isolated from the fractions of conditioned and fresh media. miR-122 enrichment in exRNA (n = 4 GSC cultures) was calculated pre- and post-correction to the levels in fresh media, as described in Supplementary Fig. 6. miR-122 was highly enriched in the GSC exRNA, relative to its intracellular level, with up to 1500-fold enrichment in the exosomes before media correction. miR-122 enrichment in GSC exosomes became marginal upon correction. All bars represent mean ± SEM comparative quantitative assessment of cellular vs extracellular mRNA or non-coding RNA. Although in three out of four GSC RNAs. Since the proportion of total small RNA, and speciﬁc types cultures, cellular mRNA reads accounted for 20–35% of long of RNA is vastly different in cellular and extracellular RNA RNA libraries, the mRNA proportion in EV fractions was below (Fig. 4a and Supplementary Fig. 3), normalization to total 10%, and even lower in RNPs (Fig. 4a). The reads obtained from mapped reads is not optimal. As recently proposed , we utilized small RNA libraries were ﬁrst mapped to the most accurately spike-in RNA for normalization, and quantiﬁed the abundance of annotated miRNA database (miRBase), and subsequently to other RNA species as fmol per μg of total RNA. Further, since fresh databases with equal mapping priority. In total, all annotated culture media (FM) contain RNA that is co-isolated with cell- RNA species were categorized into 14 classes (Fig. 4a). Since small derived exRNA , we also assessed the interference of FM RNA RNA libraries were built on 15–65 nt transcripts, the vast with downstream analysis of GSC exRNA. With this goal, we majority of the mapped reads represent fragments rather than isolated three RNA fractions from the corresponding FM, using full-length transcripts, with exception of miRNA and piRNA the same ﬁltration procedure, and subjected them to RNAseq in reads. Despite the remarkable heterogeneity of the GSC cultures, parallel with the GSC exRNA. Approximately 1 ng/ml total different extracellular fractions exhibited common characteristics exRNA was isolated from FM, and it was largely associated with of their RNA repertoires. Some of the most distinct features of the RNP fraction (Supplementary Fig. 4). Overall, FM contributed GSC-derived exRNA are summarized as follows: (1) mRNA 1.3–15% to the exRNA isolated from conditioned media, varying exons and snoRNAs are depleted, compared to cellular RNA, in between the fractions (Fig. 3b). Although this amount of all extracellular fractions; (2) all extracellular fractions, especially contaminating RNA is small, it can still affect the results of non-vesicular RNPs, are highly enriched in speciﬁc Y RNA exRNA enrichment analysis. For example, miR-122 falsely fragments of largely unknown functions; (3) MVs and exosomes showed exosomal enrichment (Fig. 3c), consistent with previous differ in their RNA composition, with mRNAs being relatively 20, 21 reports , due to its abundance in B-27 supplement more enriched in MVs and miRNAs in exosomes; (4) RNP (Supplementary Fig. 5). Based on this observation, we included fractions have a highly distinctive RNA repertoire, with tRNA FM RNA data set in our RNAseq analysis pipeline, to provide the and Y RNA fragments strongly enriched, and snRNA and repeats baseline for GSC-derived exRNA (Supplementary Fig. 6). The reduced. The predominance of tRNA and Y RNA fragments in results described below were obtained using the media correction. RNP is reﬂected in the corresponding sharp ~32 nt peak observed in the length distribution proﬁle of reads (Supplementary Fig. 7). The relative abundance of piRNA and scRNA (small cytoplasmic EVs and RNPs exhibit distinct RNA composition. To char- RNA) fragments was also higher in RNP fractions. However, the most abundant individual piRNA and scRNA species were acterize the repertoire of GSC cellular and extracellular RNA, we sequenced the libraries of small and long RNAs, and ﬁrst nor- identical or highly homologous to major tRNA and Y RNA fragments, respectively (Supplementary Fig. 8). Whether such malized the number of reads for each RNA class to the total identical sequences indeed belong to two functionally distinct number of non-rRNA reads within the library, thereby removing the confounding factor of variable rRNA depletion efﬁciencies. classes of transcripts, or tRNA and Y RNA are commonly mis- identiﬁed due to the poor quality of databases, is unknown. In All reads generated on the long RNA libraries (that generally included transcripts longer than 100 nt), were classiﬁed as either addition, recently discovered circular RNAs have been reported as 4 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | RNP Exosome MV 4.0% miR-122 enrichment (ratio to cellular RNA) RNA concentration (ng/ml media) NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE Cell MV Exosome RNP Long RNA: 50 ncRNA 40 mRNA Small RNA: miRNA tRNA Y RNA 70 piRNA snRNA snoRNA SRP RNA scRNA 40 vRNA mRNA exons mRNA introns Repeat Other ncRNA 10 Other genome bd ** *** *** mRNA Full length Amplicon length NS NS NS NS * * 0.5 RPS27 361 nt 306 nt RPLP2 511 nt 451 nt 0.0 RPS6 829 nt 724 nt –0.5 EEF2 3163 nt 2996 nt 3′-UTR 5′-UTR YY1AP1 2705 nt 2509 nt CDS –1.0 MV Exosome RNP PROM1 3857 nt 3708 nt Cell MV Exosome RNP >6000 5500–6000 5000–5500 20/3 4500–5000 GBM4 4000–4500 GBM8 3500–4000 MGG75 3000–3500 2500–3000 2000–2500 1500–2000 1000–1500 500–1000 0–500 Number of transcripts Fig. 4 Relative composition of diverse RNA classes in cellular and extracellular compartments (MVs, exosomes, and RNPs) in different GSC cultures. a The top panels exhibit relative RNA composition in long RNA libraries, and the bottom panels depict the composition in small RNA libraries. The data were normalized to the total number of annotated non-rRNA reads. The bars framed in red represent the mean values of four GSC cultures. b RT-PCR analysis (with equal input of total RNA) of selected mRNAs abundant in exRNA, demonstrates the presence of nearly full-length short, but not long messages in the extracellular fractions. Long RT-PCRs were replicated twice. c Long RNA libraries-based analysis of the length distribution of 500 most abundant mRNAs suggests no length preference for shorter parent transcripts in the extracellular fractions. d Analysis of the RNAseq reads mapped to mRNAs indicates that UTR regions were more enriched than CDS regions in the extracellular fractions (n = 4 GSC cultures). mRNA reads were aligned to the coding sequences (CDS), 5′-UTRs, and 3′-UTRs separately, and the alignment rates for each extracellular fraction were normalized to the corresponding cellular fraction. The log-transformed ratios of the alignment rates were compared among the three regions. Error bars represent mean ± SEM. NS, not signiﬁcant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test 22 24, 25 enriched in exRNA , and although our RNAseq protocol was not fragments in exRNA . On average, our RNAseq detected optimized for their quantiﬁcation, several circular RNA species 17,148 intracellular mRNAs in GSCs, whereas 17,219, 11,592, and were detected (Supplementary Table 1). 11,819 mRNAs were detected in MV, exosome, and RNP frac- tions, respectively. Many of the most abundant extracellular mRNAs corresponded to relatively short transcripts, including UTR fragments are released more than ORFs and intact mRNAs for various ribosomal proteins (Supplementary Data 1). mRNAs. The length of intact cellular mRNAs varies between 350 However, some abundant reads in EVs corresponded to long and 12,000 nt, with the average around 2000 nt . Considering the mRNA transcripts, such as PROM1 and EEF2. To examine small size of EVs, the maximum length of an mRNA that could be whether intact mRNAs are present in extracellular fractions, we packaged is still an open question. Previous high-throughput designed PCR primers for selected mRNAs to provide their full- studies have not discriminated between full-length mRNAs and length ampliﬁcation. As shown in Fig. 4b, near-complete short NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 5 | | | 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200 20/3 GBM4 GBM8 MGG75 Mean Cell 20/3 MV GBM4 Exosome GBM8 RNP MGG75 Mean 20/3 GBM4 GBM8 MGG75 Mean 20/3 GBM4 GBM8 MGG75 Mean Relative abundance (%) Length range (nt) Log ratio of alignment rate to cell ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x Evenness factors of RNA species abundance distribution RNA category Cell MV Exosome RNP All long RNA 6.37±1.49 4.71±1.53 4.67±1.04 4.92±0.47 --mRNA 11.70±0.89 12.12±0.36 14.27±3.85 16.30±2.45 --ncRNA 2.59±0.72 4.26±0.89 2.89±1.76 3.81±0.40 All small RNA 3.94±0.26 3.12±0.10* 2.26±0.19** 0.91±0.15*** --miRNA 6.09±0.34 5.27±0.12 5.39±0.35 4.76±0.30* --snoRNA 16.62±1.79 17.98±0.72 16.24±1.27 9.41±2.57 11.63±0.57 8.10±0.69** 6.98±0.58** 5.99±0.99** --snRNA --SRP RNA 17.81±0.79 15.57±0.45* 12.32±1.05** 14.19±1.43 --tRNA 19.20±0.45 17.41±0.45* 11.61±0.89*** 8.48±0.45*** --vRNA 31.25±6.25 31.25±6.25 25.00±0.00 18.75±6.25 --Y RNA 11.27±0.98 8.33±0.56* 6.54±0.27** 5.88±0.38** b c Fold change of X value to estimate heterogeneity difference Cell MV GBM4 GBM8 GBM4 GBM8 RNA category Cell MV Exosome RNP 15 17 17 17 All long RNA 1 1.340 1.720 1.660 8 10 4 2 71 --mRNA 1 0.967 2.746 2.905 18 88 26 19 14 8 29 --ncRNA 1 1.792 2.137 1.998 46 45 4 0 7 4 All small RNA 1 1.868 2.651 1.624 3 5 1 3 8 9 20/3 MGG75 20/3 MGG75 --miRNA 1 1.045 0.838 1.001 Exosome RNP --snoRNA 1 2.034 1.762 2.196 GBM4 GBM8 GBM4 GBM8 --snRNA 1 2.060 1.995 4.266 54 36 76 79 --SRP RNA 1 1.073 3.520 3.069 2 615 10 41 50 611 35 67 80 89 --tRNA 1 0.423 0.916 1.889 15 1 --vRNA 1 0.692 0.068 0.958 1 5 0 5 1 7 2 11 6 --Y RNA 1 0.401 1.030 1.002 20/3 MGG75 20/3 MGG75 Fig. 5 Inequality and heterogeneity of the RNA repertoire of extracellular fractions. a Evenness factors, reﬂecting the inequality of abundance’ distribution of the indicated RNA classes in various fractions. Higher evenness factors correspond to lower inequality. The classes of tRNA, Y RNA, snRNA, and SRP RNA, but not miRNA, contributed to the increased inequality of exRNA most signiﬁcantly. Differential evenness factors of cellular and extracellular RNA suggest the selectivity of secretion (n = 4 GSC cultures). b For each RNA category, a sum of squared errors (χ value) was calculated among four GBM cultures, after normalization of each RNA species to the total number of reads in that RNA category. The χ value of each extracellular fraction was compared to the cellular fraction. Fold change of χ values higher than 1 reﬂects the increased heterogeneity. Heterogeneity either increased or decreased more than two-fold is highlighted in red and blue, respectively. c Venn diagrams depict the number of common species among 100 top abundant mRNAs, in four GBM cultures and their extracellular fractions, supporting the observation of higher heterogeneity of mRNA composition in exRNA than in cellular RNA. Signiﬁcant differences between the cellular and extracellular fractions are depicted as following: *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test mRNAs could be detected in cellular and all extracellular frac- usually account for the majority of total miRNA in a given cel- tions, but detection of long mRNAs above 1000 nt was limited to lular context . Consistent with this observation, 31 miRNA cells and MVs only. These results suggest that either long mRNAs species accounted for 80% of the total miRNA in GBM8 cells, are excluded from packaging into exosomes and RNP complexes, indicative of a diverse range of miRNA expression (or “inequal- or they are present in these fractions only as fragments. The latter ity” of miRNA levels). Even fewer, 19 miRNA species, accounted appears to be the case, since the RNAseq demonstrates a similar for 80% of the total miRNome in GBM8 exosomes, suggesting a representation of transcripts of various lengths in the intracellular higher inequality of miRNAs in exRNA. Such comparison, and extracellular compartments (Fig. 4c). These data suggest that however, relies on a randomly selected cutoff (80% used above). most exRNA reads corresponding to long mRNAs represent To compare the inequality of intracellular and extracellular fragmented transcripts. Nevertheless, amplifying long RNA from transcripts more objectively, we developed two alternative stra- low-input exRNA fractions is technically challenging and cannot tegies. The ﬁrst one is an improved version of traditional eva- be performed in a high-throughput manner, so we cannot exclude luation named the evenness factor (ε), which deﬁnes that ε%of that some long full-length mRNAs are present extracellularly. RNA species can account for (100−ε)% of total abundance. The Next, mRNA reads were aligned separately to the coding second strategy is based on the Gini coefﬁcient, commonly used sequences (CDS), 5′-UTRs, and 3′-UTRs. The UTR regions, and for evaluation of inequality in economics. Of note, although both especially 3′-UTRs, were signiﬁcantly enriched in all extracellular are objective and use no preset cutoff, higher ε and lower Gini fractions relative to CDS sequences (Fig. 4d), validating the pre- coefﬁcient correspond to a more equal representation (or lower vious observation and suggesting differential release pathways inequality). The detailed illustration of the concepts behind these for the protein-coding and regulatory sequences. evaluation systems can be found in Supplementary Fig. 9. Taking advantage of these parameters, we compared transcript inequality in cellular and extracellular fractions, either for the whole RNA Inequality of RNA representation increases in exRNA.A library or a speciﬁc RNA class. The inequality of RNA levels was relatively small number of the most abundant miRNA species similar for cellular and extracellular long RNA libraries, but 6 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | fmol input in RT fmol input in RT fmol input in RT NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE ab c Cell 15,000 50,000 RNY1 fragment GluCTC fragment 40,000 RNY3 fragment GlyGCC fragment 10,000 30,000 RNY4 fragment GlnTTG fragment 20,000 RNY5 fragment LeuCAA fragment 5000 MV 10,000 200 250 Exosome RNP 0 0 0 30 112 5′ 3′ Nucleotide position de f Y1 Y3 Y4 U2 - miR1246 –10 Y5 Y4 - 5′Y4 Y5 - 5′Y5 Glu - 5′Glu Gly - 5′Gly –2 –20 5′ 3′ miR-199a-3p miR-92a-3p 5′Y5 –4 –5 4 25 2 25 2 18 2 19 2 –6 26 2 –5 20 2 26 2 21 2 –7 2 0 22 2 –6 27 2 –1 23 2 –8 28 2 –2 24 2 –7 2 –3 –9 2 RNAseq 29 RNAseq 2 RNAseq –4 qPCR qPCR 26 qPCR –8 –10 –5 29 2 30 2 2 Cell MV Exosome RNP Cell MV Exosome RNP Cell MV Exosome RNP Fig. 6 Y RNA and tRNA fragments are abundant and enriched in exRNA. a, b Speciﬁc Y RNA and tRNA species are among the most abundant in small RNA libraries (n = 4 GSC cultures). The reads corresponding to Y1, Y4, and Y5 are highly enriched in extracellular fractions, especially in RNP (a). The reads corresponding to speciﬁc tRNAs, such as GluCTC and GlyGCC, are highly enriched in exRNA, while others (e.g. GlnTTG and LeuCAA) are not (b). c Mapping coverage of Y1 reads indicates that the Y1 is precisely processed, and mostly its 5′ fragment is present in exosomes and RNPs, as evidenced by the steep peaks corresponding to the 5′-end 30 nt. These proﬁles are distinct from the more uniform full-length coverage observed for the cellular RNA, and to a lesser extent MV RNA. Similar analysis for other Y RNAs and tRNAs is presented in Supplementary Fig. 12. d The predicted secondary structure of Y1 RNA , Copyright (1993) National Academy of Sciences, USA, and the position of its cleavage (indicated by the arrow) that produces the 5′ fragment which is highly abundant in exRNA. e Quantiﬁcation of Y RNA reads in long and small RNA libraries, demonstrates different fragment to full-length ratios in the cell and exRNA fractions (n = 3 GSC cultures; MGG75 was excluded due to very low abundance of the full-length Y RNA). The ratios are increased in extracellular fractions. f qRT-PCR analysis with primers speciﬁc to either full-lengths or fragments of several RNA species validates the enrichment of 5′ tRNA and Y RNA fragments in extracellular fractions. For each speciﬁc transcript examined, two lines represent GBM8 and 20/3 cells, respectively. g qRT- PCR analysis of selected transcripts conﬁrms the quantitative character of the RNAseq pipeline. The blue dots represent qRT-PCR Cq values, while the red dots represent the results of RNAseq quantiﬁcation in fmol. Both analyses were performed on the same set of RNA samples. Error bars represent mean ± SEM increased signiﬁcantly in small exRNA libraries (Fig. 5a and cellular and extracellular RNA species across the GSC cultures, we Supplementary Table 2). These results provide support for the ﬁrst normalized the reads to the total read number within an idea of selective RNA incorporation into different exRNA frac- individual RNA category, and then evaluated the sum of squared 2 2 tions, that is still highly debated in the ﬁeld. errors (χ value). The higher χ value reﬂects the higher diversity/ heterogeneity of GSC cultures in terms of RNA composition. As shown in Fig. 5b, relative to cellular RNA, the extracellular Heterogeneity of RNA repertoire increases in exRNA fractions. fractions were, overall, more heterogeneous in their composition Despite the genetic diversity of GSCs, cellular RNA class com- in both long and small RNA libraries, as well as for the majority position was similar among the four different GSC cultures of speciﬁc RNA classes. This phenomenon was not caused by analyzed, with the exception of MGG75 cells that expressed more technical irreproducibility of the exRNA analysis, because inde- mRNA than long ncRNA (Fig. 4a). However, the RNA repertoire pendently analyzed exosomes produced by different passages of of extracellular fractions was more heterogeneous among GSC GBM8 cells were much more concordant than the pairwise- cultures than that of cells. To estimate the heterogeneity of compared exosomes released by GSC cultures established from NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 7 | | | C el l MV Ex o s o m e RN P Ce l MV Ex o s o m e RN P Cell MV Exosome RNP Cell MV Exosome RNP Cq value Abundance (fmol/μg total RNA) Log ratio between fragment and full length Cq value Abundance (fmol/μg total RNA) Cq - Cq full-length fragment Cq value Prefer full-length Prefer fragment Sequencing depth ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x a cd ef All mRNAs: All miRNAs: 0.0 3.5 25.0 –4.0 –1.0 1.5 0.0 0.04 0.15 R =0.4954 0.1 0.01 0.1 0.001 piRNA 0.01 R =0.9169 tRNA 0.0001 0.001 Y RNA 0.0001 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 scRNA GBM4 cell GBM4 cell miRNA abundance+0.0001 abundance+0.001 vRNA snoRNA R =0.1129 mRNA exons 0.1 SRP RNA 10 snRNA 0.01 Other ncRNA 0.1 Other genome 0.001 R =0.1685 0.01 Repeat mRNA introns 0.0001 0.001 0.0001 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 GBM4 cell GBM4 cell abundance+0.0001 abundance+0.001 PCA analysis 1 1000 MV 2 2 R =0.0020 100 R =0.0260 Exosome 0.1 0.01 2 0 RNP 0.1 –1 0.001 0.01 –2 PC1 (46.7%) 0.001 0.0001 –4 –2 –2 0 –2 0.0001 0.001 0.01 0.1 1 10 0.001 0.01 0.1 1 10 –1 –1 –1 GBM4 cell GBM4 cell –2 abundance+0.0001 abundance+0.001 1 Cellular 1 –4 –2 0 2 * *** *** *** * * *** *** ** ** * * * *** *** ** ** ** *** ** *** * *** *** *** MV Exosome RNP COL1A2 PTEN EEF2 RPS6 miR-21 miR-10b GluCTC GlyGCC Y4 5′Y4 Y5 5′Y5 Fig. 7 RNA repertoire of MV most closely reﬂects cellular RNA composition. a Heat map cluster analysis of RNA classes indicates relative similarity of the composition of MVs and the source cells. The scale bar represents the percentage of non-rRNA annotated reads. b PCA analysis of RNA classes. Different fractions are marked in different colors. Within each fraction, four dots represent four GSC cultures. c, d GBM4 MV represents the cellular mRNA content closely, and much better than exosomes or RNPs, based on the correlation analysis of all mRNA species (c), and cluster analysis of the top abundant mRNAs (d). The scale bar represents mRNA abundance. e, f Extracellular miRNA composition, in general, is less reﬂective of the cellular miRNome; nevertheless, MV fraction still remains the best simulator, based on the correlation analysis of all miRNA species (e), and cluster analysis of the top abundant miRNAs (f). The scale bar represents the log-transformed miRNA abundance. Similar analyses of other cell cultures can be found in Supplementary Figs. 14–17. g qRT-PCR analysis of exRNA fractions isolated from the CSF of GBM patients, using the same ﬁltration-based procedure, indicates its applicability to clinical samples. The data verify preferential association of selected RNA species with different exRNA fractions in the human bioﬂuid (n = 4 CSF samples). All bars represent mean ± SEM. *, p < 0.05; **, p < 0.01; ***, p < 0.001; t-test different patients (Supplementary Fig. 10). Consistently, Venn tRNA species are highly abundant in extracellular fractions, diagram analysis showed less commonalities (or increased het- especially the RNP, reaching the quantities of up to dozens of erogeneity) for mRNAs in exosomes and RNPs, relative to cellular pmol per μg of total RNA (Fig. 6a, b). Relative to let-7b-5p, one of RNA (Fig. 5c). The heterogeneity of miRNA class, however, was the most abundant miRNAs in exRNA, those molecules are at similar between GSC cellular and exRNA compartments (Sup- least hundred times more abundant. Since human Y RNAs are plementary Fig. 11). Combined analyses of inequality and het- 84–113 nt and tRNAs are 68–176 nt, and our libraries were erogeneity suggest that GSC cultures may utilize various sorting constructed from 15 to 65 nt RNA, we reasoned that the reads mechanisms for exRNA release. represent fragments rather than full-length transcripts. Notably, the coverage analysis of the reads mapped to Y RNAs and tRNAs suggested the presence of speciﬁcally processed fragments (Fig. 6c and Supplementary Fig. 12), with the processing sites located Y RNA and tRNA fragments are abundant and enriched in within the loop domains that are known to bind several pro- exRNA. The reads corresponding to tRNA and Y RNA species 28–30 teins (Fig. 6d and Supplementary Fig. 12). Further integra- constitute a signiﬁcant proportion of the rRNA-depleted small tion of the small and long RNA data sets revealed that the ratios exRNA libraries (8.5% in cells, 13.5% in EVs, and 67.5% in RNPs, of fragment to full-length Y RNAs differed signiﬁcantly among Fig. 4a). All four human Y RNA (Y1, Y3, Y4, and Y5) and some 8 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | PC3 (13.0%) Cell PC2 (17.7%) % of total exRNA MV from CSF Exosome RNP GBM4 RNP GBM4 exosome GBM4 MV abundance+0.0001 abundance+0.0001 abundance+0.0001 Top abundant mRNAs: Cell MV Exo RNP GBM4 RNP GBM4 exosome GBM4 MV abundance+0.001 abundance+0.001 abundance+0.001 Top abundant miRNAs: Cell MV Exo RNP Percentage NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE GBM MV vs HA GBM exosome vs HA GBM RNP vs HA –5 –4 –5 –4 –4 miR-196a-5p –3 miR-196a-5p miR-196a-5p miR-423-5p miR-615-3p –3 –3 miR-615-3p miR-378a-3p miR-10b-5p miR-10b-5p –2 miR-615-3p miR-378a-3p miR-10b-5p miR-378a-3p –2 –2 miR-182-5p miR-183-5p miR-182-5p miR-182-5p miR-183-5p –1 miR-199a/b-3p –1 –1 miR-378c –2 –1 0 1 2 3 –3 –2 –1 0 1 2 3 –4 –3 –2 –1 0 1 2 3 Mean log (FC) Mean log (FC) Mean log (FC) 10 10 10 Total in Total in miRNA hAst hNG HBEC mAst mMG mNeu MBEC TCGA GBM TCGA Ctrl p value human mouse miR-182-5p +++ – +++ 6 +++ +++ +++ – 9 7.823±0.064 6.789±0.054 0.024 miR-196a-5p +++ + + 5 NA NA NA NA NA 6.474±0.033 5.688±0.005 9.0E–04 miR-10b-5p +++ + – 4 +++ +++ +++ – 9 8.663±0.063 6.681±0.006 1.1E–05 miR-183-5p + – ++ 3 + +++ + – 5 6.497±0.037 6.024±0.010 0.078 miR-378a-3p +++ – –3 – – – – 0 5.929±0.004 5.908±0.011 0.524 miR-199a-3p + + – 2 – + + ++ 4 7.705±0.051 6.786±0.108 0.011 miR-199b-3p ++– 2 – + + + 3 NA NA NA miR-615-3p + + – 2 NA NA NA NA NA 6.098±0.019 5.886±0.018 0.127 miR-92b-3p – – + 1 – +++ – + 4 8.187±0.030 6.818±0.050 7.0E–10 miR-378c +–– 1 – – –– 0 NA NA NA Positive z-score z-score = 0 Negative z-score No activity pattern available Percentage 15.0 12.5 10.0 7.5 5.0 2.5 0.0 de 1.5 ** ** * * ** ** 1.0 0.5 40 Mono-culture Mono-culture Co-culture Co-culture 0.0 Timp3 Rassf9 Ptgfr Hmgb3 Hapln Cep110 U6 miR-21 Fig. 8 Comparative analysis of GSC-secreted miRNAs and cellular miRNome of the normal cells of the brain predicts the most impactful GBM miRNAs in tumor-to-microenvironment communication. a Extracellular fractions of GSC cultures were compared to primary human astrocytes (HA), based on the corresponding RNAseq data sets. The fold-changes in miRNA levels were log transformed and a t-test was applied to examine the signiﬁcance of difference. MiRNAs with log-fold changes higher than 1.7, which corresponded to 50 times higher levels in the GSC-derived exRNA fractions relative to the recipient cells, and p < 0.05 (t-test), were deﬁned as potentially impactful (colored in red). The horizontal axis of Volcano plots shows the log-fold difference, and the vertical axis shows the statistical signiﬁcance. Similar analyses of primary human neuroglial and endothelial cells are shown in Supplementary Fig. 18. b A full list of most impactful GSC miRNAs for human and mouse astrocytes, neurons, microglia, or brain endothelial cells. The number of “+” symbols reﬂects the number of extracellular fractions in which an miRNA meets the indicated criteria as in a. Most of these miRNAs are also upregulated in the GBM tumors compared to non-neoplastic brain tissues in the TCGA microarray data set, as indicated in the three right columns (n = 496 GBM vs 10 control). c Top enriched IPA pathways for the validated mRNA targets of the most impactful miRNAs. Predicted activation and inhibition of pathways are labeled as orange and blue bars, respectively. The yellow line shows the percent of genes in each pathway that are validated targets. d, e Co-cultured with GBM8 neurospheres, primary miR-21-null astrocytes exhibit steady miR-21 levels (d) and downregulation of validated miR-21 targets (e). Cq value of miR-21 in mono-cultures was deﬁned as 45 (undetectable expression). N = 4 wells in 24-well plate. All bars represent mean ± SEM. *, p < 0.05; **, p < 0.01; t-test NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 9 | | | Molecular mechanisms of cancer Glucocorticoid receptor signaling ERK/MAPK signaling AMPK signaling Breast cancer regulation by stathmin1 B-cell receptor signaling ERK 5 signaling Protein ubiquitination pathway Epithelial adherens junction signaling PI3K/AKT signaling Hypoxia signaling in the cardiovascular system NGF signaling GNRH signaling PPARα/RXRα activation HGF signaling Prostate cancer signaling CD27 signaling in lymphocytes Role of PKR in interferon induction and antiviral response Small cell lung cancer signaling Apoptosis signaling Cardiac hypertrophy signaling Regulation of elF4 and p7056K signaling CDK5 signaling Log (p) Cq value –Log(p-value) Log (p) Fold change (normalized to Actb) Log (p) 10 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x the Y RNA species (Fig. 6e). Overall, the fragment to full-length detected in CSF MVs. The preference for full-length Y RNAs in ratio was signiﬁcantly higher in extracellular vs cellular fractions CSF MVs, and their fragments in RNPs was also consistent with for all four Y RNA species (Fig. 6e), indicating that whereas full- the observations made on GSC-derived exRNA. length Y RNAs are highly abundant in the cells and MVs, speciﬁc fragments of them are enriched in the exosomes and RNPs. These Exosomal miRNAs with high functional potential upon data were further conﬁrmed by qRT-PCRs (Fig. 6f). Moreover, transfer. A common, but still highly debated, hypothesis is that such preference for fragments in the exosomes and RNPs was not EVs and EV-associated exRNA are taken up by recipient cells and limited to Y RNA. For example, full-length U2 snRNA (RNU2-1), mediate intercellular communication. MiRNA transferred from which also serves as a precursor for miR-1246 , and speciﬁc GBM to cells in their microenvironment could have signiﬁcant tRNAs are also released to a lesser extent in extracellular fractions impact on the transcriptome of the peritumoral cells. We rea- than their corresponding processed products (Fig. 6f). As indi- soned that the greatest impact would be mediated by those cated by the experiment with unfractionated exRNA, fragmen- miRNAs that are most abundant in EVs/exRNA, while not or tation is a feature of exRNA rather than an artifact of the minimally expressed in recipient cells, such that their transfer ﬁltration procedure (Supplementary Fig. 13). Of note, qRT-PCRs would signiﬁcantly alter their levels in recipient cells and thus quantiﬁcation of the selected transcripts correlated well with the affect the regulation of mRNA targets. To identify such miRNAs, RNAseq results (Fig. 6g), supporting the accuracy of our RNAseq we compared relative levels of all miRNAs in exRNA fractions analysis. from GSCs with those in the major types of human and mouse brain cells, including astrocytes, mature neurons, microglia, and MVs most closely reﬂect cellular RNA composition. Cancer- brain-derived endothelial cells. Comparison of three GSC extra- derived exRNA may serve as clinical biomarkers for disease cellular fractions with primary astrocytes is shown in Fig. 8a, and diagnostics, prognostics and monitoring. An extracellular fraction similar analysis for other cell types is presented in Supplementary that closely mirrors the deranged cellular transcriptome would be Fig. 18. Summary of this analysis, performed on three extra- the most valuable for such applications. To date, mostly unfrac- cellular fractions and four types of recipient cells, revealed a list of tionated, precipitated or 100,000 g-pelleted EVs have been the GBM-derived miRNAs that might potentially have the explored as a source for potential biomarkers. To examine what strongest impact on the normal cells of brain microenvironment type of extracellular complexes (MVs, exosomes, or RNPs) might (Fig. 8b). In agreement with these data, most of the listed serve as the closest proxy of the GSC transcriptome, we per- miRNAs were also found to be elevated in GBMs relative to the formed correlation and clustering analysis of their RNA com- control brain tissues in The Cancer Genome Atlas (TCGA) data position. Overall, clustering analysis based on small RNA libraries set (Fig. 8b). To predict the downstream effects of the 10 most demonstrated the highest similarity between the cellular tran- impactful miRNAs on the recipient cells, we analyzed their direct scriptome and MV content, and to a lesser extent to exosome mRNA targets previously validated by at least three supporting content, while the RNP fraction had a highly distinct RNA CLIP-Seq data sets in the starBase database . In total, 2267 composition (shown for GBM4 in Fig. 7a). This was further mRNA species interact with at least one of the impactful miR- conﬁrmed by principal component analysis (PCA) of all four NAs. Based on the ingenuity pathway analysis (IPA), these targets GSC cultures (Fig. 7b). Similar analyses of mRNA and miRNA are signiﬁcantly enriched in many canonical cancer-related classes, and the most abundant transcripts within them, further pathways and bioterms, including the molecular mechanism of supported this conclusion (Fig. 7c–f and Supplementary Figs. 14– cancer, ERK/MAPK signaling, PI3K/AKT signaling, and NGF 17). Consistent with this idea, there were fewer miRNA species, signaling (Fig. 8c). and overall fewer different RNA species signiﬁcantly enriched in MVs than in exosomes and RNPs (Supplementary Data 2). The Less than one copy per EV on average for most RNA species.A observation that MVs provide a more accurate peripheral read- substantial amount of transferred exRNA might be required to out of the source cell content is in line with recent extracellular exert a functional effect in a recipient cell; however, the quanti- proteomic analysis . Therefore, MVs which include large vesicles tative data for the levels of speciﬁc RNA classes and individual (200–800 nm) appear to be a good source for RNA biomarker transcripts in exRNAs is very limited thus far. To address this discovery, and have the potential to outperform the more studied issue, we performed a stoichiometric analysis of the smaller exosomes. EV-associated RNA. On average, GSC EVs (MVs and exosomes collectively) provided ~8.9 ng total RNA per ml of conditioned Analysis of exRNA complexes in cerebrospinal ﬂuid.To media. Considering the concentration 2 × 10 EVs per ml, mea- investigate the differential potential of exRNA fractions for bio- sured by NTA, one EV contained ~4.45 ag total RNA, or ~0.445 marker discovery, we tested our sequential ﬁltration method on ag non-rRNA, which corresponded to ~836 ribonucleotides. cerebrospinal ﬂuid (CSF) samples obtained from four GBM Similar analysis was performed for individual RNA classes and patients. As shown in Fig. 7g, sufﬁcient amounts of total RNA for species. As shown in Table 1, rRNA and snRNA are qRT-PCR analysis were isolated from each CSF fraction (MVs, present in more than one copy per EV on average. RNA classes exosomes, and RNPs), and distinct proﬁles observed for different represented by approximately one copy per EV include Y RNA RNA classes. Although we could not examine the resemblance of and Y RNA fragments, tRNA fragments, lncRNA, and miRNA. CSF exRNA proﬁles to those of parental tumor and neural cells One copy of mRNA or mRNA fragment can be found in ~10 EVs. due to unavailability of tumor material from the corresponding Of note, quantiﬁcation of individual RNA species revealed that patients, the data indicated that, indeed, MVs and exosomes only several speciﬁc molecules are present at the level of one copy should be analyzed separately in biomarker discovery studies. per EV (e.g., RNY1, Y5 fragments, GluCTC tRNA fragment, U1 Similarly to the results obtained on cultured GSC-derived exRNA, and U2 snRNA fragments). The most abundant miRNA species the CSF mRNAs were preferentially associated with MVs and (e.g., let-7b, miR-21) were present at the level of one copy per 10 miRNAs with exosomes, suggesting that MV enrichment is EVs approximately, consistent with the previous report . The warranted for mRNA biomarkers, while exosomes represent a most abundant individual mRNA species were present at one superior source for miRNA biomarkers. Of note, several mRNAs copy per 1000 EVs approximately (e.g., TMSB10), and the levels frequently mutated in GBM, including PTEN and COL1A2, were of mRNAs most commonly mutated in GBM (e.g., EGFR, IDH1, 10 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE TP53, PTEN, and COL1A2) were not higher than one copy per required to affect signaling or alter the phenotype of the recipient 100,000 EVs approximately. Based on these data, continuous cells via their exRNA content. barrage, massive and/or highly selective uptake of EVs might be To examine the functionality of transferred GSC-derived miRNAs, we established 3D transwell co-cultures of GBM8 neurospheres with primary mouse astrocytes, a system more physiological than the commonly used exposure of recipient Table 1 Quantiﬁcation of selected RNA classes and cultures to super-concentrated EVs isolated from donor cell individual species in copy number per EV conditioned medium. In line with the low copy number of individual miRNAs per EV, we were unable to detect signiﬁcant Copy level RNA class or individual species elevation of either miR-21 or miR-10b (top GBM-promoting More than 1 copy per EV RNA class rRNA fragment miRNAs) in astrocytes co-cultured with GBM8. However, Repeat fragment miR-21 became readily detectable in primary astrocytes estab- snRNA fragment lished from miR-21 knockout mice upon their co-culture with One copy per 1 EV RNA class tRNA fragment GBM8 (Fig. 8d). Furthermore, this latter miR-21 transfer led to approximately functional effects in these cells, i.e., repression of its previously Intron fragment validated mRNA targets (Fig. 8e) . These data support inter- Y RNA fragment SRP RNA fragment cellular transfer of miRNA via exRNA and suggest that, despite miRNA the apparently low levels in EVs, miRNAs may exert regulatory Exon fragment functions in recipient cells, albeit with speciﬁc conditions and lncRNA highly sensitive methodologies required for detection. Y RNA snoRNA fragment Individual RNA U2 snRNA fragment Discussion species exRNA studies have expanded rapidly and multidirectionally in U1 snRNA fragment this decade. Revealing the composition of exRNA complexes GluCTC tRNA fragment released by deﬁned cell types remains one of the most funda- RNY1 mental milestones toward understanding the role of exRNA in RNY5 fragment intercellular communication, as well as discovery of RNA bio- One copy per 10 EVs RNA class mRNA markers for disease. This study provides the ﬁrst minimally approximately biased quantitative analysis of the exRNA released by tumor- vRNA derived cells from GBM patients. Despite the high heterogeneity vRNA fragment of GSC cultures established from different tumors, and even Individual RNA RNY4 fragment higher heterogeneity of the exRNA they release, extracellular species complexes share key characteristics. The RNA proﬁles of MVs, RNY4 RNY1 fragment exosomes, and RNPs are highly distinct. They all display selec- let-7b-5p tivity in their RNA loading compared with the cells, with the MVs GlyCCC tRNA being the most like the cells and the RNPs the least. Small and fragment fragmented RNAs account for the majority of exRNA in all three miR-21-5p types of complexes. Between 64 and 93% of all exRNA consists of vRNA1-2 fragmented rRNA (Fig. 3a), although there is little-to-no intact vRNA1-2 fragment rRNA (Supplementary Fig. 3). miRNA, the most studied class of One copy per 100 EVs Individual RNA U3 snoRNA fragment exRNA, constitutes <10% of non-rRNA exRNA (Fig. 4a). Con- approximately species sistent with some previous publications , and in contrast to miR-1246 20, 37–41 others , we found that miRNA species are relatively enri- miR-10b-5p RNY5 ched in exosomes, but not in MVs or RNPs. Of note, the isolation vRNA1-1 fragment approaches, the library preparation strategies, and the normal- vRNA1-3 fragment ization methods utilized in these different studies were diverse, vRNA1-3 making the results not directly comparable. Other non-coding One copy per 1000 EVs Individual RNA RNY3 fragment RNA species are more abundant, and some of them are enriched approximately species in EVs or RNPs. Notable among them are precisely processed TMSB10 mRNA (top tRNA and Y RNA fragments, associated with both EVs and 1 mRNA) extravesicular RNPs, and supported by observations of other miR-93-5p 20, 37 42 cultured cells and body ﬂuids . One copy per 10,000 EVs Individual RNA ACTB mRNA approximately species The most common extracellular 30–32 nt-long 5′-tRNA frag- GAPDH mRNA ments (5′-tRFs), also called 5′-tRNA halves or tiRNAs, are evo- miR-132-3p lutionarily conserved molecules produced by angiogenin One copy per 100,000 EVs Individual RNA COL1A2 mRNA 44 (ANG), a member of ribonuclease A family . This multi- approximately species functional ribonuclease regulates angiogenesis, cell proliferation RNY3 and viability of cancer cells, as well as neuronal survival and stress miR-34a-5p response . Speciﬁc5′-tRFs are known to perform crucial func- IDH1 mRNA tions, often associated with regulation of gene expression in stress EGFR mRNA response. They repress protein translation by displacing eukar- 1 copy per 1,000,000 EVs Individual RNA TP53 mRNA yotic translation initiation factors eIF4E and eIF4G ; and mod- approximately species PTEN mRNA ulate stress response by inducing formation of stress granules— cytoplasmic foci where untranslated mRNAs are transiently The transcripts were deﬁned as fragments if present and quantiﬁed in small RNA libraries stored . Importantly, speciﬁc5′-tRFs (e.g., 5′Ala, NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 11 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x 5′His, and 5′Cys) selectively regulate translation of subsets of nuclear proteins PCNA and Ro60 appeared enriched in RNPs but mRNAs, both capped and uncapped, and therefore reprogram not in EVs. While the co-localization of Ro60 with its binding Y protein synthesis . They may also function in a miRNA-like RNA partners in RNPs may point to the functionality of this 46 47, 48 manner . ANG is upregulated in GBM and is one of the extracellular complex, the role of extracellular PCNA, a protein proteins most highly secreted by glioma cells . Despite its high involved in DNA replication, remains to be elucidated. Although abundance, functions of ANG, including its extracellular activity the possibility of direct non-vesicular, RNP-mediated RNA and its exRNA products remain to be investigated. Remarkable uptake and function in recipient cells is currently unexplored, it is 61, 62 enrichment of both ANG and 5′-tRFs observed in GSC-derived supported by the utility of RNPs for RNA and drug delivery . exosomes (Figs. 2b and 6f) suggests that tRNA cleavage may Regardless of the potential biological function of RNPs, their occur in exosomes, outside of the cells. associated RNAs expand the repertoire of potential biomarkers Another highly abundant but poorly studied classes of that should be further explored in body ﬂuids, in parallel to EV non-coding exRNAs are Y RNAs and their speciﬁc5′ fragments. transcripts. The majority of information about Y RNA has come from studies The key outcome of our work, overall, is an expansion of the of bacteria and invertebrates. In vertebrates, Y RNAs are repertoire of exRNAs released by GSCs in different vehicles, with expressed in all tissues and cells and have been proposed to functional and biomarker potential, far beyond the class of 50, 51 participate in many important cellular functions . Their lower miRNA. This conclusion challenges the commonly assumed stem domain, which recruits chaperone Ro60 and exoribonu- predominant role of miRNA in exRNA-mediated intercellular clease PNPase, may play a role in RNA quality control and communication. It further points to the need for in-depth degradation of misfolded RNAs . The upper stem domain may investigation of other classes of exRNAs and their impact on the participate in initiation of chromosomal DNA replication . With physiology of recipient cells and use as biomarkers. The future biogenesis and activity independent of Dicer and Ago2 , Y RNA should bring the development of novel experimental techniques fragments do not appear to silence gene expression in a miRNA- and computational resources for integrating complex expression like manner . Recent studies reported that Y RNA fragments data sets into comprehensive biologic networks and biomarker may be involved in histone mRNA processing and cell discovery. damage . Interestingly, Y5 fragment, the most abundant in exRNA, is proposed to specialize in surveillance of ribosomal Methods RNA . Studying the functions of these precisely processed, GBM stem cell cultures. Human low-passage (below 20) GBM cells (kind gift from Dr. Hiroaki Wakamoto, MGH) were cultured as neurospheres in Neurobasal highly enriched extracellular transcripts represents an exciting medium (Gibco) supplemented with 3 mM GlutaMAX (Gibco), 1× B-27 supple- new avenue in RNA biology. ment (Gibco), 0.5× N-2 (Gibco), 20 ng/ml EGF (R&D systems, MN), 20 ng/ml FGF In addition to non-coding RNA, we detected low levels of (PEPROTECH, NJ) and 0.5% Antibiotic-Antimycotic Solution (Corning), and mRNA reads with UTRs relatively enriched compared to the passaged by NeuroCult Chemical Dissociation Kit (Mouse) (Stemcell Technologies, Canada) following the manual. Approximately 5 × 10 dissociated cells were seeded coding regions in exRNA. The mechanism underlying this per 10 cm dish (Corning) in 10 ml fresh media, and 1/3 volume of fresh medium enrichment and its biological impact has yet to be investigated. was added every 3 days. Mature neurospheres, typically formed in 7–10 days, were 58, 59 Methylation and other RNA modiﬁcations of UTRs might dissociated and replated. All cells were tested for mycoplasma. Human cells were cause differential sorting or stability, leading to enrichment in used in accordance with the policies of institutional review boards at Brigham and Women’s Hospital. exRNAs. UTRs and their fragments might function as molecular sponges for various regulatory molecules, including miRNA, translation factors (e.g., eIF4F and ribosomal complexes), other Primary cultures of normal brain cells. Brain cortices of E18 and P1 C57BL/6 mice were dissected for primary cultures of neurons and glial cells, respectively. RNA-binding proteins, and thereby exert functions in recipient The tissues were dissociated with 0.25% Trypsin (Gibco) and 0.1 mg/ml DNaseI cells. Altogether, our data indicate the strong preference of pro- (Roche) for 15 min at 37 °C. The cells were plated in poly-D-lysine-coated T25 cessed fragments for multiple classes of RNA, protein-coding and 2 ﬂasks or 24-well plates at ~80,000 cells per cm , in the seeding medium consisting non-coding, in exRNA (Fig. 6). The co-packaging of of DMEM-F12 (Corning), 10% FBS (Gibco), and 1% Antibiotic-Antimycotic Solution (Corning). For neuronal cultures, the media was exchanged to Neurobasal processed RNA with various RNA-binding proteins, including (Gibco), 2% B-27 (Gibco), 1% Antibiotic-Antimycotic Solution (Corning) and 0.5 RNases (e.g., ANG) and effecter complexes (e.g., Ago2), and the mM Glutamax (Gibco) 1 day after plating. Mature neurons at 21 days in culture signiﬁcant depletion of full-length RNA in exosomes, suggests have been utilized for the RNAseq. For glial cultures, the ﬂasks were shaken (200 that they may function as an exRNA processing machinery with rpm at 37 °C) three times overnight to remove microglia, and astrocytes trypsinized and further cultured in 24-well plates. For microglia cultures, the media was specialized autonomous functions. supplemented with the recombinant M-CSF mouse protein (10 ng/ml; Gibco). Although several studies have explored the RNA bound to Floating microglial cells were collected from the conditioned media by gentle spin speciﬁc secreted or circulating proteins, such as high-density (300×g, 10 min), and re-plated in 24-well poly-D-lysine-coated plates at ~100,000 3 60 lipoprotein (HDL) and Ago2 , overall little attention has been cells per cm . For human primary neuroglial cultures, fetal cortical tissues paid thus far to the non-vesicular exRNA complexes that account (gestational age 18 weeks) were provided by Advanced Bioscience Resources, Inc. (Alameda, CA), dissociated with papain (12 U/ml; Worthington), seeded with for the signiﬁcant proportion of exRNA (Fig. 2c). We present data neuronal media plus 2% FBS (Gibco), and cultured as mouse neurons . Neuronal indicating that extracellular non-vesicular RNPs exhibit a RNA cultures at 30 days in vitro have been utilized for the RNAseq. Human (HBEC) and signature readily distinguishable from EVs. Notably, ncRNA mouse (MBEC) primary brain microvascular endothelial cells were purchased from accounts for nearly entire RNA population in RNPs, with a large Cell Biologics, IL (Catalog# H-6023; Lot# 021514F14 and Catalog# C57-6023; Lot# 070613T2MP, respectively) and cultured accordingly to the manufacturer. All prevalence of tRNA, Y RNA and their products, and depletion of animal experiments have been approved by the Harvard Medical Area Standing miRNA species (Figs. 4 and 6), consistent with the analysis of Committee on Animals. breast cancer extracellular RNPs . Although extracellular RNPs might be highly heterogeneous, this collective data suggest the key Fractionation and RNA isolation. Approximately 100 ml conditioned medium pathways involved in the biogenesis. Of note, we found that was used as input for exRNA isolation. Conditioned media was centrifuged at massive levels of albumin, an abundant component in B-27 300×g, 4 °C for 10 min, following by the additional centrifugation at 2000×g,4°C for 15 min, to remove cells and cell debris. To monitor EVs, the samples were supplement, hamper the protein characterization of extracellular diluted in DPBS and examined using the Nanoparticle Tracking Analysis system RNPs, and markers of RNPs have yet to be deﬁned. Beneﬁting (NanoSight LM10; Malvern Instruments, UK), and EV concentrations were from an efﬁcient (>95%) albumin-depletion protocol, we were quantiﬁed within the optimal linear range (2–10 × 10 particles per 1 ml). able to compare protein content in MVs, exosomes, and For RNA preparation, 5 μl of SUPERase In RNase Inhibitor (Ambion) was non-vesicular RNPs using immunoblots. Interestingly, two added to the supernatants per 10 ml media. The media was then ﬁltered 12 NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications | | | NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x ARTICLE sequentially through the 2 μm ﬁlter (GE Healthcare, UK), 0.8 μm ﬁlter (EMD performed by Cuffdiff with parameter -u/--multi-read-correct and normalized Millipore, MA), and 0.22 μm ﬁlter (EMD Millipore), with no/minimal pressure based on the ERCC spike-in controls using the cyclic loess robust local regression. applied. The ﬁltrate was split to 15 ml per sample and further ﬁltered through the Clean reads produced from the small RNA libraries were ﬁrst aligned to carrier 0.02 μm ﬁlter (GE Healthcare) with up to 75 psi pressure applied. To facilitate the RNA (Enterobacteria phage MS2) of miRCURY Spike-in kit with bowtie 0.02 μm ﬁltration, a mechanical syringe pump was designed and manufactured (parameter: -v 2 -l 7 --all). The unmapped reads were aligned against human rRNA (Supplementary Fig. 2). Upon ﬁltration, each ﬁlter was washed with 1 ml DPBS sequences with bowtie (parameter: -v 2 -l 7 --all) to remove the remaining rRNA (Corning), and the corresponding fractions were lysed with 600 μl lysis solution of reads. The remaining reads were mapped to human miRNA precursors (miRBase the miRCURY RNA Isolation Kit—Cell & Plant (Exiqon, Denmark). The fractions V19) with bowtie (parameter: -v 1 --all), and the miRNA precursors reads were collected on 0.02 μm ﬁlters were lysed with 900 μl lysis solution. The last ﬂow- separated according to the mapping position. The mapped reads with more than through fractions of 0.02 μm ﬁlters were pooled together (up to 30 ml) and 10% mismatched nucleotides were excluded. The remaining reads were mapped to concentrated ~60 times using 3 kDa Amicon Ultra Centrifugal Filters (EMD spike-ins (UniSp2, UniSp4 and UniSp5; parameter: -v 1 --all), and other non- Millipore) at 4000×g, 4 °C, for 60 min. The concentrates were collected and lysed coding RNA classes including tRNAs (gtRNAdb), piRNAs (RNAdb), snoRNA with six volumes of the same lysis solution (Exiqon). Total RNA was then isolated (snoRNA-LBME-db), scRNAs (Genbank), and others (Rfam database) (parameter: from all fractions as recommended by miRCURY protocol, with -v 2 --all). Finally, the remaining reads were mapped to the hg19 human genome, on-column DNase treatment (Qiagen, Germany). The corresponding 1.2 ml of the and the reads mapped to exons, introns, intergenic regions, and repeats (parameter: source neurospheres were span down at 300×g, 4 °C for 5 min, and total cellular -v 0 --all) identiﬁed. The SAMtools was used to calculate the reads depth for each RNA was isolated from them and analyzed in parallel. The same protocol was base position and R package barplot was used to draw the corresponding bar plot. carried out for RNA isolation from fresh media, with 500 ml media input. For RNA isolation from primary cells cultured in 24-well plates, the cells were lysed with 350 μl lysis solution per well. The concentrations of cellular and extracellular RNA were Data analysis. Based on the known amounts of spike-in RNAs, the read count for determined using the NanoDrop 2000 Spectrophotometer and Quant-iT each RNA species was ﬁrst normalized to spike-ins’ reads, to quantify the absolute RiboGreen RNA Assay Kit (Thermo Fisher Scientiﬁc), respectively. The RNA amounts in fmol per μg of total RNA. Next, the obtained values were corrected to quality was examined using Agilent 2100 Bioanalyzer (Agilent, CA) and the RNA the corresponding fresh media as the blank control, using the equation shown in Integrity Number (RIN) estimated. Supplementary Fig. 6. For the analysis of class composition, the total abundance of corrected non-rRNA was used for normalization between the samples. To compare heterogeneity of the samples, the sum of squared errors (χ value) of species Transmission electron microscopy. The material collected on the ﬁlters was composition was calculated using MS Excel Macro (available in Supplementary resuspended using DPBS (Corning), and further pelleted by 100,000×g UC for 80 Data 3). To estimate the inequality of abundance among all RNA species in one min at 4 °C. The material diluted in DPBS was added to a glow-discharged carbon- class, the evenness factor, Gini coefﬁcient, and traditional pre-set evaluations were coated grid. The grids were washed with distilled water, stained with 0.75% uranyl calculated using a MS Excel Macro (available in Supplementary Data 4). The formate, examined using Tecnai G Spirit BioTWIN microscopy (FEI, OR), and hierarchical clustering analysis was performed using the MultiExperiment Viewer images recorded by the AMT 2k CCD camera (Advanced Microscopy Techniques, (Dana-Farber Cancer Institute, MA). The principle component analysis (PCA) was MA) at the Harvard Medical School EM Facility. performed and visualized with R package rgl. miRNA targets were determined based on starBase v2.0, with at least three supporting CLIP-Seq experiments . RNA sequencing. Two sets of spike-in RNAs were added to the samples prior to Pathway analysis of target mRNAs was performed using the Ingenuity Pathway library preparation: ERCC RNA Spike-In Control Mixes (Ambion; 0.02 μl per 1 μg Analysis (IPA; Qiagen). Venn diagrams were plotted using Venny 2.1. total RNA), and miRCURY Spike-in kit, part 1, with UniSp2’s ﬁnal concentrations of 1.25 and 5.0 fmol per 1 μg of total RNA for cellular and extracellular RNA, respectively. Total RNA, either 40–200 ng of exRNA, or 2 μg of cellular RNA, was Long RNA reverse transcription PCR. Maxima Reverse Transcriptase (100U; rRNA-depleted using the Ribo-Zero rRNA Removal Kits (Illumina, CA). One Thermo Fisher Scientiﬁc) was used to reverse transcribe 20 ng RNA with both oligo quarter of the rRNA-depleted RNA was fragmented to 100–500 nt using the 5× (dT) and random hexamers in a 10 μl reaction system. Next, 0.5 μl cDNA was used First-Strand Buffer (Clontech, CA), and utilized for the long RNA library con- in 10 μl PCR reactions based on Phire Hot Start II PCR Master Mix (Thermo struction by SMARTer Stranded RNA-Seq Kit (Clontech). The remaining 75% of Fisher Scientiﬁc) and primers (0.5 μM each), and ampliﬁed using a touchdown the rRNA-depleted RNA was treated sequentially with Tobacco Acid Pyropho- program with either 15 or 70 s extension step for short or long ampliﬁcation, sphatase (TAP; Illumina) and T4 Polynucleotide Kinase (T4PNK; New England respectively. The PCR products were examined on 0.8–1.2% agarose gels (Thermo Biolabs, MA) to create more uniform 5′- and 3′-ends for various classes of tran- Fisher Scientiﬁc). Primer sequences are provided in Supplementary Table 3. scripts. The RNA was then used as input for the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs), with size selection of 15–65 nt inserts for small RNA libraries. The quality of libraries was examined using the Quantitative reverse transcription PCR. Generally, for small RNA qRT-PCR, 10 Agilent DNA 1000 kit at the Agilent 2100 Bioanalyzer instrument, and cDNA ng of total RNA was used in 10 μl reverse transcription reaction with Universal quantiﬁed by qRT-PCR. The libraries were sequenced on HiSeq 2000 (Illumina) cDNA Synthesis kit II (Exiqon). The cDNA was diluted 80 times, and 4 μl was used with single read 50 cycles by Beijing Genomics Institute (BGI, China). in 10 μl qPCR reactions using the ExiLENT SYBR Green master mix and custom- designed LNA primers (Exiqon). For mRNA qRT-PCR, 10 ng of total RNA was used in 10 μl reverse transcription reaction with PrimeScript RT Master Mix Reads annotation. Sequencing reads were treated using the BGI pipeline that included multiple ﬁltering steps, as follows: (1) removing reads with adapters; (2) (Takara, Japan). One microliter cDNA was used in 10 μl qPCR reactions using the ExiLENT SYBR Green master mix and synthesized primers (Supplementary removing reads with >10% of unknown bases; and (3) removing low-quality reads Table 4; Eton Bioscience, CA). The qPCR reactions were run on a ViiA 7 (sequencing quality <10). After ﬁltering, the remaining clean reads were subjected instrument (Thermo Fisher Scientiﬁc) in duplicates. The speciﬁcity of qPCR to the bioinformatics analysis. Generally, 20 and 10 M clean reads were generated products was veriﬁed by the presence of a single peak at the melting curves. per long RNA library and small RNA library, respectively. The clean reads pro- duced from the long RNA libraries were ﬁrst mapped to the Homo sapiens rRNA database using the SOAPaligner/SOAP2 short read alignment software, to remove the remaining rRNA reads. The non-rRNA reads were used to perform the tran- Immunoblotting. For protein analysis, cellular and extracellular fractions were scriptome assembling and quantiﬁcation. First, non-rRNA reads were mapped to lysed using the modiﬁed RIPA buffer containing 2% SDS, 1% sodium deoxycholate human reference genome hg19 using an improved version of TopHat2, which and 3 M urea. The RNP fractions were BSA-depleted using the Aurum Afﬁ-Gel aligns reads across splice junction without relying on gene annotation. Default -g/-- Blue Mini Columns (Bio-Rad, CA), and concentrated using Pierce SDS-PAGE max-multihits was used to allow up to 20 multimapping. Next, the reads mapped Sample Prep Kit (Pierce, MA). Protein concentrations were quantiﬁed using the to genome were assembled using Cufﬂinks. Reference Annotation Based Transcript Micro BCA Protein Assay Kit (Pierce). Equal amounts of the total protein (50 μg) (RABT) assembly was performed with the reference gene annotation to compen- were loaded per lane in Novex WedgeWell 14% Tris-Glycine Mini Gel (Thermo sate incompletely assembled transcripts caused by read coverage gaps in the regions Fisher Scientiﬁc). Proteins were transferred to 0.45 μm PVDF membrane (Thermo of reference gene. The set of transfrags generated was then compared with the Fisher Scientiﬁc). After blocking with 5% (wt/vol) fat-free milk in Tris-buffered reference transcripts to remove transfrags that were approximately equivalent to saline with 0.075% Tween-20 (TBST), membranes were incubated with 1:1000 the whole or a portion of a reference transcript. After the assembling, the whole diluted primary antibodies (Flotillin-1 #18634S, CD9 #13174S, Integrin β1 #9699S, parsimonious set of transcripts was obtained. These transcripts were blasted with HSP90 #4874S, La #5034S, NPM #3542S, Ago2 #2897S, Alix #2171S, and PCNA the NONCODE database using the ﬁlter set (identity >0.9 and coverage >0.8) to #13110S from Cell Signaling Technology, MA; Ro60 #AV40534 from Sigma- identify known long non-coding RNA. Then, the rest of assembled transcripts were Aldrich; ANG #sc-74528 from Santa Cruz Biotechnology, TX) overnight at 4 °C. aligned to the reference annotation utilizing Cuffcompare. Thereafter, Cuffmerge The membranes were washed and incubated with horseradish was utilized to merge several assemblies from different samples together, which peroxidase–conjugated secondary antibodies (#7074S and #7076S from Cell Sig- automatically ﬁltered out a number of transfrags that probably were artifacts and naling Technology, 1:2000 dilution) for 1 h at room temperature. The blots were produced a single annotation ﬁle for downstream gene expression analysis. Two developed by the Amersham ECL Reagent (GE Healthcare) and, if required, mismatches were allowed for annotation generally, and only one mismatch was stripped using One Minute Western Blot Stripping Buffer (GM Biosciences, CT). allowed for the ERCC spike-ins. mRNA and lncRNA expression analyses were Uncropped scans of blots are shown in Supplementary Fig. 19. NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 13 | | | ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-01196-x Transwell cocultures of GSC and astrocytes. Mouse cortical astrocytes estab- 14. Teplyuk, N. M. et al. Therapeutic potential of targeting microRNA-10b in lished from WT or miR-21 KO P1 pups, passage 1 or 2, were cultured in established intracranial glioblastoma: ﬁrst steps toward the clinic. EMBO Mol. 24-well plates. 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We thank Robert Kitchen, Sai Lakshmi Subramanian, Alain Charest, Steven associate with AGO proteins to recognize speciﬁc RNA targets. BMC Biol. 12, Gould, Thorsten Mempel, and the RNAseq working group of the Extracellular RNA 78 (2014). Communication Consortium for valuable insights on the data analysis. We thank Maria 47. Eberle, K. et al. The expression of angiogenin in tissue samples of different Ericsson for TEM, Hongjun Wang for designing and manufacturing syringe pumps, brain tumours and cultured glioma cells. Anticancer. Res. 20, 1679–1684 (2000). Pavel Ivanov for sharing ANG antibody, Oleg Butovsky for sharing miR-21 knockout 48. Xia, W. et al. Angiogenin promotes U87MG cell proliferation by activating NF- mice, and Erik J. Uhlmann for reviewing the manuscript. We thank members of Kri- kappaB signaling pathway and downregulating its binding partner FHL3. PLoS chevsky and Breakeﬁeld laboratories for helpful discussions. ONE 10, e0116983 (2015). 49. Skog, J. et al. 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Genes Dev. 29, 1998–2003 (2015). Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in 56. Chakrabortty, S. K., Prakash, A., Nechooshtan, G., Hearn, S. & Gingeras, T. R. published maps and institutional afﬁliations. Extracellular vesicle-mediated transfer of processed and functional RNY5 RNA. RNA 21, 1966–1979 (2015). 57. Hogg, J. R. & Collins, K. Human Y5 RNA specializes a Ro ribonucleoprotein for 5S ribosomal RNA quality control. Genes Dev. 21, 3067–3072 (2007). Open Access This article is licensed under a Creative Commons 58. Meyer, K. D. et al. Comprehensive analysis of mRNA methylation reveals Attribution 4.0 International License, which permits use, sharing, enrichment in 3′ UTRs and near stop codons. Cell 149, 1635–1646 (2012). adaptation, distribution and reproduction in any medium or format, as long as you give 59. Ke, S. et al. A majority of m6A residues are in the last exons, allowing the appropriate credit to the original author(s) and the source, provide a link to the Creative potential for 3′ UTR regulation. Genes Dev. 29, 2037–2053 (2015). Commons license, and indicate if changes were made. The images or other third party 60. Arroyo, J. D. et al. Argonaute2 complexes carry a population of circulating material in this article are included in the article’s Creative Commons license, unless microRNAs independent of vesicles in human plasma. Proc. Natl Acad. Sci. indicated otherwise in a credit line to the material. If material is not included in the USA 108, 5003–5008 (2011). article’s Creative Commons license and your intended use is not permitted by statutory 61. Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efﬁcient RNA-guided regulation or exceeds the permitted use, you will need to obtain permission directly from genome editing in human cells via delivery of puriﬁed Cas9 ribonucleoproteins. the copyright holder. To view a copy of this license, visit http://creativecommons.org/ Genome Res. 24, 1012-1019 (2014). 62. Zhang, F. et al. Reconstituted high density lipoprotein mediated targeted licenses/by/4.0/. co-delivery of HZ08 and paclitaxel enhances the efﬁcacy of paclitaxel in multidrug-resistant MCF-7 breast cancer cells. Eur. J. Pharm. Sci. 92,11–21 © The Author(s) 2017 (2016). NATURE COMMUNICATIONS 8: 1145 DOI: 10.1038/s41467-017-01196-x www.nature.com/naturecommunications 15 | | |

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Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

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Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

Coding and noncoding landscape of extracellular RNA released by human glioma stem cells

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