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Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions

Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of... 9272–9285 Nucleic Acids Research, 2012, Vol. 40, No. 18 Published online 19 July 2012 doi:10.1093/nar/gks658 Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions 1, 2 1 Esther N. M. Nolte-’t Hoen *, Henk P. J. Buermans , Maaike Waasdorp , 1 1 2 Willem Stoorvogel , Marca H. M. Wauben and Peter A. C. ’t Hoen Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht and Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands Received March 14, 2012; Revised June 13, 2012; Accepted June 14, 2012 INTRODUCTION ABSTRACT Nano-sized membrane vesicles represent a recently Cells release RNA-carrying vesicles and membrane- identified class of intercellular communication vehicles free RNA/protein complexes into the extracellular operating in many organisms (1–6). Such vesicles can milieu. Horizontal vesicle-mediated transfer of derive from multivesicular bodies (MVBs), which are such shuttle RNA between cells allows dissemin- late endosomal compartments containing multiple ation of genetically encoded messages, which may 50–100 nm sized intraluminal vesicles. Fusion of MVBs modify the function of target cells. Other studies with the plasma membrane causes the release of their used array analysis to establish the presence of intraluminal vesicles, which are then called exosomes (7). microRNAs and mRNA in cell-derived vesicles Alternatively, vesicles can be released by cells through direct shedding from the plasma membrane (1). Cells from many sources. Here, we used an unbiased can tightly regulate the release and molecular composition approach by deep sequencing of small RNA of these vesicles (8,9) and vesicle targeting depends on the released by immune cells. We found a large variety type and activation status of recipient cells (10,11). of small non-coding RNA species representing per- Despite their early description decades ago (12,13), the vasive transcripts or RNA cleavage products wide-spread occurrence of cell-derived vesicles and their overlapping with protein coding regions, repeat se- potential for tailor-made modulation of target cell quences or structural RNAs. Many of these RNAs behaviour has only been recognized during the last few were enriched relative to cellular RNA, indicating years. It is now clear that cell-derived vesicles are not that cells destine specific RNAs for extracellular only released by almost all cultured cell types, but are release. Among the most abundant small RNAs in also present in a wide range of body fluids (1). Since the molecular make-up and release of cell-derived vesicles is shuttle RNA were sequences derived from vault regulated by the producing cell, these vesicles are of RNA, Y-RNA and specific tRNAs. Many of the interest for disease-related biomarkers (14). Moreover, highly abundant small non-coding transcripts in extracellular vesicles may be used as therapeutic agents shuttle RNA are evolutionary well-conserved and (15,16). have previously been associated to gene regulatory Besides specific sets of lipids and proteins, cells can functions. These findings allude to a wider range of shuttle RNA into vesicles determined for release into the biological effects that could be mediated by shuttle extracellular space. This allows the conveyance of genet- RNA than previously expected. Moreover, the data ically encoded messages between cells (17). The first key present leads for unraveling how cells modify the publication on nucleic acids in cell-derived vesicles function of other cells via transfer of specific reported the presence of miRNA and mRNA in vesicles non-coding RNA species. derived from mast cells and the functional transfer of *To whom correspondence should be addressed. Tel: +31 30 2534336; Fax: +31 30 2535492; Email: [email protected] The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2012, Vol. 40, No. 18 9273 RNA to vesicle-targeted cells (17). More recently, the MATERIALS AND METHODS luminal protein and RNA contents of cell-derived Cell culture vesicles were demonstrated to be delivered into the cyto- D1 cells were maintained in Iscove’s Modified Dulbecco’s plasm of recipient cells via fusion of vesicles with these medium supplemented with 2 mM Ultraglutamine cells (18). It is currently not known whether all vesicle (Biowhittaker), 10% heat inactivated fetal calf serum populations released by cells contain RNA. Although (FCS, Sigma-Aldrich), 100 IU/ml penicillin and 100 mg/ several studies indicated that the RNA composition of ml streptomycin (GIBCO), 50 mM b-mercaptoethanol cell-derived vesicles is different from the parental cell and 30% conditioned medium from GM-CSF producing (17,19–21), it is unknown how RNAs are selected for se- NIH 3T3 cells (R1). The p53-specific CD4 T-cell clone, cretion into the extracellular space. Furthermore, extracel- generated in a C57BL/6 p53–/– mouse (34) and provided lular RNA may also be associated with macromolecular by Prof C. Melief (Leiden University Medical Center, complexes that are not enclosed by a vesicle. We here Leiden, The Netherlands), was cultured as described pre- collectively refer to extracellular RNA as ‘shuttle RNA’. viously (8). For cognate DC/T cell co-culture conditions, Although the majority of circulating miRNAs in human DC were pre-loaded with 2.5 mM p53 peptide (amino acid plasma and serum were found to co-fractionate with 77–96) for 2 h and subsequently mixed in a 1:1 ratio with T proteins such as Argonaute2 (Ago2) and nucleophosmin, cells, and co-cultured for 20 h in medium containing over- which have been suggested to protect miRNAs from deg- night ultracentrifuged (100 000g) FCS and R1 conditioned radation in RNase-rich environments (22,23), it is cur- medium (to deplete bovine and R1 derived vesicles and rently unknown if vesicle-free RNAs can bind and large protein aggregates). All cultures were maintained modify the function of target cells. at 37 C, 5% CO . Cell death in DC and T cell cultures Next-Generation Sequencing (NGS) techniques have was less than 5% and cognate DC-T cell interactions did led to the discovery of large numbers of unexpected not lead to additional cell death in the indicated co-culture non-coding RNAs (24). These transcripts were found to period. Experiments were approved by the institutional overlap with exons, introns and intergenic regions (24–30). ethical animal committee at Utrecht University (Utrecht, Various non-coding transcripts found in close vicinity to The Netherlands). protein coding genes, such as promoter-associated long and short RNAs, transcription start site-associated Shuttle RNA isolation RNAs, and PROMoter uPstream Transcripts (PROMPTs) are suspected to act as regulatory elements 15  10 p53 peptide-pulsed DCs were co-cultured with 15  10 T cells in 25 ml culture medium as described to modulate gene activity (30). Interestingly, many small earlier. Supernatants were pooled to a total of 450 ml. non-coding RNA species have been found that could act This culture supernatant was subjected to differential cen- as regulatory RNAs similar to miRNAs. Fragments trifugation (35). In short, cells were removed by two se- derived from small nucleolar RNA (snoRNA), vault quential centrifugations at 200g for 10 min. Collected RNA (vRNA) and transfer RNA (tRNA), for example, supernatant was subsequently centrifuged two times at were shown to bind Argonaute (AGO) proteins and form 500g for 10 min, followed by 10 000g for 30 min. Vesicles RNA-induced silencing complexes (RISCs) capable of and protein/RNA complexes were finally pelleted by ultra- regulating expression of target mRNAs analogous to centrifugation at 100 000g for 65 min in SW28 and SW40 miRNA-containing RISCs (31–33). rotors (Beckman). When indicated, the 100 000g sedi- The analysis of shuttle RNA populations has almost mented material was further fractionated using gradient exclusively focused on miRNAs and mRNAs, most density centrifugation. To this end, the 100 000g sedi- likely due to the availability and ease of array hybridiza- mented material was mixed with 2.5 M sucrose, overlaid tion techniques to detect these RNAs. Which other small with a linear sucrose gradient (2.0–0.4 M sucrose in PBS) RNA biotypes were released by cells via vesicles or in and floated into the gradient by centrifugation in a SW40 protein complexes was unknown. We therefore aimed to tube (Beckman) for 16 h at 192 000g. The bottom two frac- comprehensively analyze small RNA species released by tions (1.26–1.28 g/ml, as measured by refractometry) and cells using NGS. Among the most well studied cell-derived fractions with densities of 1.12–1.18 g/ml were pooled, vesicles are those produced by dendritic cells (DCs). These diluted with PBS/0.1% BSA and centrifuged again at cells are the sentinels of the immune system and the inter- 100 000g for 65 min. Small RNA (<200 nt) was isolated action of DC with T cells is the key event in initiation of from 100 000g pellets and from co-cultured DC and T immune responses. We previously found that DC-T cell TM cells (15  106 each) using the mirVana miRNA interactions induce vesicle release by DC and that Isolation Kit (Ambion) according to the manufacturer’s DC-derived vesicles can be targeted to activated T cells procedure. The RNA integrity and size distributions were (8,11). The release and targeting of RNA-containing assessed using Agilent 2100 Bioanalyzer pico-RNA chips. vesicles by DC and T cells may play an important role in the communication between these cells and the Small RNA sequencing library generation ensuing immune response. Here, we extensively characterized small (<70 nt) shuttle RNA released Sequencing libraries were generated as described (36). In during DC-T cell interactions and compared this RNA short, small RNA fractions were ligated to adaptors using with small cellular RNA in order to investigate selectivity the SOLiD Small RNA Expression Kit (Ambion). in the extracellular release of RNA species. Libraries were pre-amplified using primers containing 9274 Nucleic Acids Research, 2012, Vol. 40, No. 18 sequences that make the SREK libraries compatible with 5p mmu-mir-191 forward: 5 -CGGAATCCCAAAAGCA the Illumina flow cell. DNA was denatured for 30 at 98 C GCTG 00  00  00 All forward miRNA primers were used in combination followed by 18 cycles with 30 at 98 C, 30 at 65 C, 30 at with the NCode universal reverse primer. 72 C and final extension for 5 at 72 C. Library fragments Y-RNA forward: 5 -GTGTTTACAACTAATTGATCA were separated on a native 6% gradient pre-cast PAGE CAACC gel (Novex, Invitrogen). The 100–150 bp size fractions Y-RNA reverse: NCode universal reverse primer containing inserts of 20–70 nt in length were excised, SRP-RNA forward: 5 -GGAGTTCTGGGCTGTAGT DNA was eluted from the gel, precipitated and dissolved GC in nuclease free water. DNA yield was quantified using an SRP-RNA reverse: 5 -ATCAGCACGGGAGTTTTG Agilent Bioanalyzer high sensitivity DNA chip. Small AC RNA inserts were single end sequenced for 35 cycles using standard Illumina protocols. Cycling conditions were as follows: 95 C for 10 min followed by 50 cycles of 95 C for 10 s, 57 C for 30 s and Small RNA sequence data analysis 72 C for 20 s. All PCR reactions were performed on the Bio-Rad iQ5 Multicolor Real-Time PCR Detection Adaptor trimming, genome alignment and small RNA an- System (Bio-Rad). Raw threshold cycle (Ct) values were notation were performed using the E-miR data analysis calculated using the iQ5 Optical System software v.2.0 pipeline as described (36). Sequence reads were aligned to using automatic baseline settings. Thresholds were set in the Mouse Mm9 reference genome with bowtie, allowing the linear phase of the amplification plots. for alignments to three loci with two mismatches, followed by a crossmapping correction as described by de Hoon et al. (37). For further annotation with coding and RESULTS repeat RNAs, reads with a minimum overlap of 1 nt were clustered into regions and regions containing a Sample preparation and sequencing total of more than five reads in shuttle and cellular Specific antigen-bearing DCs were co-cultured with RNA were annotated using custom perl scripts (38) with cognate T cells, leading to mutual activation of both cell 0 0 exon, 3 -UTR, 5 -UTR, intron and RNA biotype informa- types. Vesicles and large protein complexes released tion from Ensembl biomart (39), all in a strand-specific during these interactions were isolated from cell culture manner. Intersections with repeat regions were determined supernatant by differential centrifugation, with a final pel- using information from the repeat track in the UCSC leting step at 100 000g (35). Total RNA was isolated from genome browser (NCBI137/mm9) and the Galaxy inter- this fraction and we observed that the majority of this face, considering a minimum of 1 nt overlap between the shuttle RNA consisted of small RNAs (<200 nt), with sequenced and the repeat region. To determine reproduci- minor amounts of 18S and 28S rRNA (Figure 1A). bility of the sample preparation and sequencing protocols, Next, small (<200 nt) RNAs were isolated from the we have evaluated the Pearson’s correlation between the 100 000g sedimented material, and processed for analysis abundance of small RNAs in replicate experiments. A by NGS. To assess whether selective RNAs were released square root transformation on the number of counts per into the extracellular space, small RNA from cells that small RNA was applied to stabilize variance and reduce had released the shuttle RNA was analyzed in parallel. influence of high-abundant small RNAs on the Pearson’s Strand specific small RNA sequencing libraries were correlation coefficient (38). prepared and pre-amplified as described (36). Fragments with inserts between 15 and 70 nt were gel purified, after Quantitative real-time PCR which the first 35 bases of the library were read with a single read, with the Illumina platform. Fragments 70 nt Sequencing derived expression profiles were compared were not included to prevent the large number of tRNAs with qPCR measurements on small RNA fractions from (70 nt in size) present in the cellular RNA fraction to independent biological samples. cDNA was generated dominate the sequencing reaction, which would hamper using the Ncode miRNA First-Strand cDNA Synthesis quantitative comparison of the cellular and shuttle RNA kit (Invitrogen). An equivalent of 100 pg RNA was used fractions. Sequencing data were processed using a in the PCR reactions with 100 nM primers (Isogen) and modified version of the E-miR pipeline (36). The se- 4 ul SYBR Green Sensimix (Bioline) in an 8 ml reaction. quences were mapped to the mouse genome (NCBI137/ PCR amplification efficiencies were determined using mm9), using bowtie allowing for two mismatches and 10-fold dilution series of template DNA and were three alignments per sequence read, followed by a between 1.8 and 2.3 for all primer sets. Transcript crossmapping correction (37). specific forward primers for miR-29a, miR-155 and To validate the robustness of our sequencing results, we miR-191 were based on full-length miRbase sequences replicated the presented experiment with a slightly differ- (40): ent sample preparation method (including an extra RNA 3p mmu-mir-29a forward: 5 -TAGCACCATCTGAAAT fragmentation step). We found strong concordance when CGGTTA ranking the different RNAs based on their abundance in 5p mmu-mir-155 forward: 5 -GGGTTAATGCTAATTG the shuttle RNA. We calculated the correlation between TGATAGGG the (ranked) small shuttle RNA sequencing data of the Nucleic Acids Research, 2012, Vol. 40, No. 18 9275 [nt] Cellular RNA Shuttle RNA Mt_rRNA Mt_tRNA miRNA misc_RNA rRNA snRNA snoRNA cellular RNA shuttle RNA 7SK RNA SCARNA SRP-RNA Vault RNA Y-RNA Figure 1. Shuttle RNA mainly consists of small RNA species and contains relatively low amounts of miRNA. Cell culture supernatant of DC-T cell co-cultures was subjected to several differential centrifugation steps, followed by RNA isolation from the 100 000g sedimenting material (shuttle RNA). (A) Bioanalyzer electropherogram of total shuttle RNA derived from DC-T cell co-cultures. The composition of small (<200 nt) shuttle and cellular RNA was further analyzed by deep sequencing. (B) Sequence reads uniquely aligned to the genome were annotated to known non-coding RNA transcripts as annotated in Ensembl using the E-miR pipeline. Data represent the percentages of reads in the indicated categories of annotated transcripts in shuttle RNA (grey bars) versus cellular RNA (black bars) calculated relative to the total sum of reads for Ensembl non-coding RNA transcripts. (C) Distribution of shuttle and cellular RNA transcripts over the different RNA biotypes within the Ensembl-annotated miscellaneous RNA (misc_RNA) category. Data are represented as the percentages of annotated transcripts as in (B). replicates. A high correlation was observed between the RNAs over the different biotypes. We found that the replicates (Pearson’s correlation 0.97, P-value <2.2e-16; majority of sequences present in the cellular small RNA Supplementary Figure S1). population represented miRNAs (Figure 1B), which is in agreement with previous studies (36,41). In contrast, the percentage of miRNAs in shuttle RNA was very low. miRNAs are underrepresented in shuttle RNA compared Instead, this fraction contained a relatively higher percent- with cellular RNA age of small ribosomal RNA (rRNA) and RNA belonging An overview of the sequencing data analysis and results to the miscellaneous category (misc_RNA) (Figure 1B). can be found in Figure 2. We found that 8.3  10E Within this category of misc_RNA, we found that struc- cellular and 27.5  10E shuttle RNA sequences aligned tural SRP-RNA, vRNA and Y-RNA were most abundant to the mouse genome (Figure 2). Uniquely aligned in shuttle RNA (Figure 1C). sequence reads were annotated to known non-coding small RNA transcripts as annotated in Ensembl. The miRNA distribution in shuttle and Remarkably, the percentage of sequence reads mapping cellular RNA is different to this category of RNAs was much lower in shuttle RNA released from cells (0.5%) compared with cellular Since the initial discovery of miRNAs in cell-derived RNA (31%). Within this category of Ensembl-annotated vesicles (17), many research groups have studied the oc- non-coding small RNAs, we analyzed the distribution of currence of this type of regulatory RNAs in vesicles % of reads % of reads 9276 Nucleic Acids Research, 2012, Vol. 40, No. 18 Truncated sequences Cells: 9,887,963 Shuttle: 28,820,290 Aligned sequences Cells: 8,273,374 (83.7%) Shuttle: 27,500,560 (95.4%) Annotation EmiR pipeline Annotation exons, introns, and repeats Ensembl small Not Ensembl small Aligned and in regions with > 5 non-coding RNA non-coding RNA reads Cells: 2,568,765 (31.0%) Cells: 5,704,609 (69.0%) Cells: 4,880,148 (59.0%) Shuttle: 134,932 (0.49%) Shuttle: 27,365,628 (99.5%) Shuttle: 18,063,486 (65.7%) miRNA not miRNA Exons Introns Repeats Cells: 1,471,703 (57.3%) Cells: 1,097,062 (42.7%) Cells: 1,967,028 (40.3%) Cells: 512,776 (10.5%) Cells: 1,453,876 (29.8%) Shuttle:10,784 (7.8%) Shuttle:124,148 (92.2%) Shuttle: 1,255,824 (6.7%) Shuttle: 4,946,811 (27.4%) Shuttle: 3,502,030 (19.4%) Exons of protein Exons of non-protein coding regions coding regions Cells: 967,229 (49.2%) Cells: 999,799 (50.8%) Shuttle: 1,060,780 (84.5%) Shuttle: 195,044 (15.5%) Figure 2. Overview of the sequencing data analysis and results. Indicated are the number of reads in each analysis step for small cellular versus shuttle RNA and the relative amount of reads calculated as a percentage of the reads detected in the previous steps. derived from various cell types (as listed in the ExoCarta process, but that cells actively sort selective miRNAs for database (42)). Although miRNAs formed only a small extracellular destination. minority within the total pool of shuttle RNA sequences Shuttle and cellular RNA contain different sets of (Figure 1B), we examined whether a specific set of transcripts miRNAs was released during DC-T cell interactions. The miRNAs occurring in cells of the DC-T cell co-culture Since the shuttle RNA population contained only 0.5% and shuttle RNA released by these cells were ranked based Ensembl-annotated small non-coding RNA transcripts, on the E-miR data and the 10 most prevalent miRNAs in we next aimed to define which other types of transcripts each population were compared (Table 1). miRNAs were were present in this population. By comparison with 0 0 named according to the 5 (5p) or 3 (3p) arm of the genomic locations obtained from Ensembl Biomart (39), hairpin where they originated from (36). Certain we found that 26.1% of the shuttle RNA reads (against miRNAs, such as miR-29a and miR-31, were abundant 49.9% in cellular small RNA) mapped to introns and in both cellular and shuttle RNA. However, several highly exons (Figure 2). Cellular small RNAs showed a higher abundant cellular miRNAs, such as miR92a-1 and let-7b, frequency of exonic localization (including a large number occurred only in very low abundance in shuttle RNA. of exons coding for miRNAs). However, the relative Conversely, the highly abundant shuttle miRNAs amount of exonic sequences mapping to protein coding miR-223, miR-142 and miR-93 were less abundant in regions was higher in shuttle RNA. In addition, shuttle cellular RNA. These data indicate that dissemination of RNA contained relatively more intronic sequences miRNAs into the extracellular space is not a random (Figure 2). Data have recently accumulated demonstrating Nucleic Acids Research, 2012, Vol. 40, No. 18 9277 Table 1. Enriched miRNAs in cellular and shuttle RNA that protein-coding genes can also produce a complex set of non-coding RNAs (43), including transcripts miRNA miRNA arm Rank Rank in originating from introns and from the untranslated in cells shuttle RNA regions (UTRs) (30,43). Interestingly, we observed that Abundant cellular miRNAs the percentage of reads in protein coding loci that 0 0 miR-155 5p 1 29 aligned to 3 - and 5 -UTRs was much higher in shuttle miR-29a 3p 2 1 RNA compared with cellular RNA (Figure 3A). Most miR-92a-1 3p 3 860 of these transcripts had the same directionality as the miR-31 5p 4 5 miR-15b 5p 5 46 coding mRNA. Although their exact function is miR-744 5p 6 47 unknown, these UTR-derived small RNAs have been sug- miR-let-7b 5p 7 119 gested to play a regulatory role in the attenuation or regu- miR-191 5p 8 4 lation of translation (44). miR-24-2 3p 9 10 miR-24-1 3p 10 12 Further analysis of the exonic sequences in shuttle and Abundant shuttle miRNAs cellular RNA revealed that the large majority (84%) of miR-29a 3p 2 1 exonic reads in shuttle RNA annotated to protein miR-21 5p 15 2 coding transcripts (Figure 3B), preferentially locating in miR-223 3p 41 3 miR-191 5p 8 4 the UTR regions of those transcripts (Figure 3A). Also the miR-31 5p 4 5 relative abundance of the small non-coding vRNA, miR-142 5p 47 6 Y-RNA and SRP-RNA was higher in shuttle RNA miR-93 5p 48 7 compared with cellular RNA. In contrast, lincRNA and miR-103-1 3p 18 8 miR-103-2 3p 14 9 miRNA are less abundant in shuttle RNA compared with miR-24-2 3p 9 10 cellular RNA (Figure 3B). Taken together, these results suggest that shuttle RNA is enriched in non-coding RNAs miRNAs were ranked according to the frequency of their occurrence other than miRNAs and lincRNAs and that many of these (number of reads). Indicated are the 10 most prevalent miRNAs in cells or shuttle RNA and the corresponding ranking in the other sample. are still to be annotated. Cellular RNA Shuttle RNA sense sense antisense antisense 15 15 3'-UTR 5'-UTR 3'-UTR 5'-UTR cellular RNA 45 shuttle RNA Figure 3. Exonic hits in shuttle RNA preferentially locate to UTR regions. (A) Exonic hits were analyzed for overlap with UTR regions. Indicated is the percentage of reads in shuttle RNA (left) and cellular RNA (right) mapping to the sense or antisense strand of UTR-regions, calculated relative to the total number of reads in Ensembl annotated exons. (B) The identity of exonic hits in shuttle (black) and cellular (grey) RNA. Indicated are the percentages of reads in the different RNA biotypes calculated relative to the total number of exonic reads. lincRNA snoRNA miRNA processed transcript protein coding pseudogene rRNA snRNA snoRNA Vault Y R A - N SRP-RNA 7SK -RNA % of exon reads % of exonic reads in prot coding regions 9278 Nucleic Acids Research, 2012, Vol. 40, No. 18 cellular RNA shuttle RNA cellular RNA shuttle RNA Figure 4. Different distribution of RNA repeat sequences in shuttle and cellular RNA. Abundance and classification of sequencing reads that match different types of RNA repeats. Indicated are the percentages of all reads (A) or reads in the top 1000 of most abundant regions (B) in the indicated repeat types, as detected in shuttle (black) and cellular (grey) RNA. The distribution of RNA repeat sequences is different in repeat sequences (Figure 4B). The most prevalent types of shuttle and cellular RNA repeats in shuttle RNA were tRNAs, LTRs, LINEs and simple repeats. LTRs, LINEs and simple repeats were Since the majority of small shuttle RNA transcripts did relatively much more abundant in shuttle RNA not correspond to Ensembl-annotated small non-coding compared with cellular RNA (Figure 4B). or protein-coding RNAs, we next investigated whether the as yet undefined transcripts corresponded to Specific tRNA fragments are abundantly and selectively repeat-derived RNAs. The relative abundance of repeat present in shuttle RNA RNAs in shuttle and cellular RNA was approximately the same (+/ 25%, Figure 4A). However, the distribu- To investigate in greater detail the most abundant se- tion over the type of repeat sequences in these two RNA quences in shuttle RNA, all unique regions were ranked populations was different. Shuttle RNA contained rela- by abundance (number of reads found per region) and the tively large numbers of Long Interspersed Elements RNA biotypes of the top 75 highest ranked hits were (LINEs), Long Terminal Repeats (LTRs) and simple analyzed. For comparison, a similar top 75 ranking was repeat sequences, whereas the majority of repeat se- made for the sequences found in cellular RNA (Table 2). quences in the cellular RNA consisted of tRNAs We observed that specific snoRNA and miRNAs were (Figure 4A). To further classify the most abundant among the most abundant sequences in the cellular repeat-associated RNA species, we next considered only small RNA fraction, but not in shuttle RNA. However, the top 1000 of most prominently present shuttle and as expected from previous data (Figure 4B), the shuttle cellular RNA. The differences in the distribution of the RNA was more abundant in different types of repeat se- most abundant shuttle and cellular RNA were much more quences. The high abundance of tRNA hits in both the pronounced. Almost 90% of the 1000 most abundant se- shuttle and cellular RNA fractions was remarkable, since quences in shuttle RNA (versus 33% in cells) consisted of we restricted our sequencing analysis to <70 nt RNAs. tRNA scRNA snRNA rRNA srpRNA SINE tRNA LINE scRNA LTR n A simple repeats s RN low complexity repeats rRNA non repeats srpRNA SINE LINE LTR simplerepeats low complexity repeats non repeats % of reads in regions top 1000 % of total reads Nucleic Acids Research, 2012, Vol. 40, No. 18 9279 Consequently, the sequence reads were not likely to rep- shuttle RNA, fragments of tRNA-Asp-GAY were only abundant in cellular RNA. We observed that the resent mature full-length tRNAs. We next explored majority of abundant tRNA hits in shuttle RNA whether these reads could represent tRNA fragments covered larger regions of about 40–50 nt (Table 3 and (tRFs), which have recently gained interest due to their Figure 5), caused by the presence of reads representing suspected regulatory nature (45). tRNA fragmentation is tRNA fragments (rather than full-length tRNAs) a specific process, with the composition, abundance and missing the first 5–15 nt of the mature tRNA. cleavage site varying per organism and cell type. Remarkably, at the same genomic locations, the Fragments of 18–22 nt, 30–35 nt and 50 nt locating at 0 0 coverage in cellular RNA was restricted to 30–35 nt frag- the 3 -or5 - end of mature tRNAs have previously been ments (Figure 5), indicating the presence of two different described (45–47). In cellular RNA, we observed frag- 0 0 fragments of the same tRNA, of which one is uniquely ments located at the 5 or 3 end of the mature tRNA present in shuttle RNA. (Table 3). However, the most abundant tRNA hits in shuttle RNA were all located at the 5 end of mature A specifically cleaved part of vRNA is abundantly present tRNAs. Differences were also observed in the type of in shuttle RNA tRNAs occurring in the top 75 most abundant hits of cellular and shuttle RNA; whereas fragments of Apart from the repeat and tRNA fragments, three other tRNA-Lys-AAA were abundant in both cellular and RNA biotypes were abundantly present in shuttle RNA (Table 2): (i) vRNA; (ii) SRP-RNA (7SL-RNA); and (iii) Y-RNA. All three RNAs are non-coding polymerase III RNA transcripts. Due to our <70 nt size restriction, we Table 2. Most abundant biotypes in cellular and shuttle RNA did not expect to find full-length transcripts of these three RNA biotype No. of reads No. of regions RNA biotypes. vRNAs are a family of RNAs found associated with the vault ribonucleoprotein complex Cellular RNA located in the cytoplasm (48). The function of these snoRNA 615 728 20 vault particles are largely unknown, but they are miRNA 599 709 20 tRNA repeat 586 583 27 thought to play a role in transportation of molecules, Unclassified 379 199 4 such as mRNA, from the nucleus to the cytoplasm and rRNA 107 855 2 in drug metabolism (e.g. in tumor cells). Mouse vRNA is Protein coding 103 877 1 141 nt long, but it was recently discovered that small vault vRNA 15 776 1 Shuttle RNA RNAs (svRNAs) can be generated from vault non-coding tRNA repeat 670 645 47 RNAs through a DICER-dependent and Simple repeat 484 945 6 DROSHA-independent mechanism (32). These svRNAs LINE 325 549 5 can downregulate expression of specific genes, similar to rRNA 120 797 5 miRNAs. Interestingly, we found that the predominant vRNA 45 546 1 Protein coding 26 492 4 coverage on the vRNA in exosomes was in only one of SRP-RNA 18 553 2 the internal stem loop structures (Figure 6A, B). In Y-RNA 5929 1 contrast, the coverage in cellular RNA was predominantly 0 0 at the 3 and 5 ends of vRNA, resembling the localization Regions were ranked by abundance (number of reads per region) and the top 75 highest ranked hits were categorized based on RNA biotype. of the described human svRNAs (32). These data indicate Indicated are the collective number of reads mapping to the different that a specifically cleaved part of the vRNA is preferen- biotypes and the number of different regions over which these reads tially shuttled into the extracellular space. were distributed. Table 3. Most abundant tRNA fragments in cellular and shuttle RNA tRNA fragment Location No. of reads No. of regions Region length (nt) Abundant cellular tRNA hits tRNA-Asp-GAY 3 -end 389 792 14 35 tRNA-Lys-AAG 5 -end 106 551 6 30–35 tRNA-Lys-AAA 5 -end 36 995 3 30 tRNA-Lys-AAG 3 -end 18 079 1 35 nt Abundant shuttle tRNA hits tRNA-Lys-AAA 5 -end 246 384 6 35 tRNA-Lys-AAG 5 -end 134 033 9 50 tRNA-Gly-GGA 5 -end 85 930 7 50 nt tRNA-Gly-GGY 5 -end 81 737 9 40 tRNA-Val-GTG 5 -end 44 160 5 50 tRNA-Glu-GAG 5 -end 27 900 5 40 tRNA-Val-GTA 5 -end 13 198 2 40–50 Hits in tRNAs in the top 75 highest ranked hits were annotated with anticodon, location of the region within the tRNA coding sequence (3 -or 5 -end), collective number of reads (accumulative for different copies of the same type of tRNA), the number of regions over which these reads were distributed, and the length of the regions. 9280 Nucleic Acids Research, 2012, Vol. 40, No. 18 Shuttle RNA Cellular RNA 5’ 3’ 3’ 5’ Coverage shuttle and cellular RNA Extra coverage shuttle RNA anticodon Figure 5. Different coverage of tRNA genes in shuttle and cellular RNA. (A) Screen shots were taken from the UCSC genome browser (http:// genome.ucsc.edu/) and show examples of tRNAs. Custom tracks were added to show sequence coverage in shuttle and cellular RNA. An example is shown of a tRNA gene (tRNA-Lys-AAG) on which differential coverage was detected in shuttle RNA (top) and cellular RNA (bottom). The y-axis represents the coverage at each genomic position. (B) tRNAscan-SE-predicted (http://lowelab.ucsc.edu/tRNAscan-SE) secondary structure of the mouse tRNA gene (tRNA-Lys-AAG) presented in (A). Indicated are the regions with coverage in shuttle and cellular RNA (dotted line), the additional coverage in shuttle RNA (black line), and the anticodon (circle). and RNAs (50). We found a 28 nt Y-RNA fragment and SRP- and Y-RNA are highly enriched in cell-derived 26–50 nt SRP-RNA covering regions in shuttle RNA. The vesicles abundance of SRP- and Y-RNA in material released by SRP-RNA (bound to the signal recognition particle) and cells into the extracellular space is remarkable, since these Y-RNA (bound to the Ro ribonucleoprotein complex RNAs belong to a small group of host RNAs that is also (49)) both function in intracellular transport of proteins selectively incorporated together with viral genomes into Nucleic Acids Research, 2012, Vol. 40, No. 18 9281 Main coverage shuttle RNA A B Main coverage cellular RNA Shuttle RNA Cellular RNA 5’ 3’ Figure 6. Different coverage of vRNA in shuttle and cellular RNA. (A) Screenshot from the UCSC genome browser showing the sequence coverage on the vRNA gene in shuttle RNA (top) and cellular RNA (bottom). The y-axis represents the coverage at each genomic position. (B) MFOLD-predicted (http://mfold.rna.albany.edu/) secondary structure of mouse vRNA at 21 C. Indicated are the regions with predominant coverage in shuttle RNA (black line) and cellular RNA (dotted lines). the capsids of several different viruses (51,52). To establish DISCUSSION a link between viruses and cell-derived vesicles regarding The complexity and diversity of the pools of extracellular incorporation of these RNAs, we investigated whether RNA in cell-derived vesicles and vesicle-free complexes is full-length SRP- and Y-RNA released in our DC-T cell daunting. Many research groups have focused on co-cultures were indeed associated to membrane vesicles. analyzing miRNAs in material released from a broad Hereto, the 100 000g material that was sedimented from range of cell types. However, the size distribution of the culture supernatant was further fractionated by shuttle RNA, as shown here and by others (53,54), gradient density ultracentrifugation. This allowed separ- extends well beyond the 20–23 nt size of miRNAs and is ation of RNA associated to large protein complexes, mostly in the range of 20 to 200 nt. This indicates that which are retained in the bottom of the gradient, and also other small RNA species are released by cells into the RNA enclosed in membrane vesicles, which float to low- extracellular space. By deep sequencing of the <70 nt buoyant densities (1.12–1.18 g/ml (35); Figure 7A). fraction of this shuttle RNA, we found a large variety of Subsequently, the presence of SRP- and Y-RNA in the small non-coding RNA species representing pervasive different density fractions was analyzed by RT-qPCR in transcripts or cleavage products overlapping with independent experiments. For comparison, the presence of protein coding regions, repeat sequences, or structural these RNAs was analyzed in the cellular <200 nt RNA RNAs. We extended this analysis with detailed evaluation fraction obtained from the DC-T cell co-culture from of the sequence coverage of several of the most abundant which the shuttle RNA was derived. Invariant endogenous RNA biotypes in shuttle RNA. This led to the discovery controls (reference genes) for qPCR analysis of that cellular and shuttle RNA can contain different small vesicle-enclosed RNA are unknown. As an alternative, RNAs derived from the same non-coding RNA, as was all samples were normalized to the total input quantity the case for tRNA and vRNA. Although no conclusions of RNA. Various miRNAs, which were relatively can be drawn regarding the absolute concentration of dif- abundant in cellular and/or shuttle RNA (Table 1), were ferent RNA species, the data allow comparison of the analyzed in parallel as a control. We observed that full- relative amounts of RNA types in the pools of cellular length SRP- and Y-RNA indeed associated to low-density and shuttle RNA. The unequal distribution of the cell-derived vesicles and not to high-density complexes. detected RNA species over cellular and shuttle RNA, Moreover, these RNAs were highly enriched in vesicles combined with increasing evidence for their role in gene compared with cells (Figure 7B). In contrast, all tested regulation strongly suggest that cells specifically release miRNAs were relatively more abundant in cells these RNAs to modify the function of target cells. compared with cell-derived vesicles, confirming the sequencing data (Figure 7C). Conclusively, SRP- and miRNA Y-RNA are highly abundant in cell-derived vesicles, similar to the enrichment of these host RNAs in virions. From both the sequencing and RT-qPCR data it became These findings may point to an evolutionary conserved clear that miRNAs were underrepresented in shuttle RNA mechanism by which cellular RNA and viral genomes compared with cellular RNA (Figures 1B and 7C). are selected and/or stabilized in membrane vesicles with However, the sequencing data revealed that the miRNA extracellular destination or by which transferred RNA can composition in shuttle RNA is not a mere reflection of the function in cells targeted by these vesicles. cellular miRNA (Table 1), indicating that a specific set of 9282 Nucleic Acids Research, 2012, Vol. 40, No. 18 Low density 1.12-1.18 g/ml High density 1.26-1.28 g/ml Y-RNA SRP-RNA 50 50 10 10 cells low dens high dens cells low dens high dens miR-191 miR-29a miR-155 1.2 1.2 1.2 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 cells low dens high dens cells low dens high dens cells low dens high dens Figure 7. Culture supernatant of DC-T cell co-cultures was subjected to consecutive differential centrifugation steps. The 100 000g pelleted material was loaded at the bottom of a sucrose gradient, after which low-density vesicles were floated to equilibrium density by ultracentrifugation. Low-density fractions (1.12–1.18 g/ml) and high-density (1.26–1.28 g/ml) bottom fractions of the gradient were collected (A). Small RNA (<200 nt) was isolated from the 100 000g pelleted material present in these fractions. (B, C) RT-qPCR analysis of Y- and SRP-RNA (B) and different miRNAs (C) in small cellular RNA (cells), 100 000g pelleted vesicles floating to low-density fractions (low dens) and 100 000g pelleted non-floating high-density material (high dens). All samples were normalized to input quantity of RNA. Relative expression levels were calculated based on the Ct values. Indicated is the fold increase ± s.d. of PCR product in floating or non-floating material relative to cells (set to 1). miRNAs is selected for extracellular release. Evidence RNA repeats exists that miRNAs associated to cell-derived vesicles are We found that repeat sequences were highly enriched in functional upon transfer to target cells (17,18). Although shuttle RNA compared with cells and that especially the the relative amounts of released miRNAs detected here LINE, LTR and simple repeats were more abundant in may appear low, significant effects on target cell function- shuttle RNA. Due to the size restriction of RNAs ing could be imposed due to efficient and specific cellular selected for sequencing, the detected transcripts do not targeting of vesicles and the capacity of miRNAs to cause represent full-length LINE or LTR. Small RNAs derived large-scale and long-term modulation of gene expression. from these repeats (repeat-associated small interference Interestingly, 5 out of the 10 most abundant shuttle RNA) have previously been shown to originate from miRNAs have validated target genes that play important various scattered regions within repeats such as SINE, roles in immune regulation. miR-93, for example, targets LINE and LTR (57,58). Interestingly, retrotransposon Stat3, which is involved in regulation of T cell responses RNA was recently found enriched in microvesicles (55), and miR-223 targets Mef2c, which is necessary for derived from tumor cells (59). It is currently unknown the transcriptional activation of interleukins during per- whether this retrotransposon RNA can be transferred to ipheral T cell activation (56). The sequencing and qPCR other cells and whether it can insert into the target studies described here have been performed on total cell genome. However, the dissemination and active tar- shuttle RNA obtained from DC-T cell co-culture super- geting of retrotransposable elements or fragments natant. Future studies will reveal which miRNAs (and thereof to other cells may be an effective strategy of other small non-coding RNAs) are released by DC or by T cells and which target cells can be modified by this cells to modify genes and regulate gene expression in shuttle RNA. other cells. Fold increase Fold increase Nucleic Acids Research, 2012, Vol. 40, No. 18 9283 A large number of sequences detected in shuttle RNA genomes into capsids of several different viruses (51,52). It mapped to tRNA loci. Full-length tRNAs were, however, is suggested that these host RNAs are encapsidated to excluded from analysis by size selection of RNAs smaller enhance virus assembly, virion stability and/or viral infect- than 70 nt. Although we cannot exclude low-level contam- ivity. Many enveloped viruses exploit the existing routes of ination with full-length tRNA, most tRNA hits will rep- membrane traffic to leave the host cell. A relationship between endosomal and/or plasma membrane routing of resent tRNA fragments (tRFs). Many recent publications viruses and cell-derived vesicles can therefore be envi- have demonstrated that tRFs are not merely degradation sioned (64). We hypothesize that these RNAs could play products but are specific cleavage products with versatile a role in the specific sorting of regulatory RNAs into functions, such as the inhibition of translation and cell-derived vesicles. Alternatively, these RNAs could sta- guidance of other RNAs (60). We here demonstrated bilize the RNA content of cell-derived vesicles or guide that the tRF compositions in cellular and shuttle RNA regulatory RNAs for efficient functioning upon release were different, indicating selectivity in released tRFs. into the cytoplasm of target cells. Earlier evidence that tRFs can be released from cells The abovementioned RNA biotypes make up only and transported to distant cells comes from the field of around 50% of the total transcripts present in shuttle plant physiology, where specific tRFs were identified in RNA. We found that many of the other transcripts the phloem sap of several different plants (61). The 5 located to intergenic regions. One example is a highly end located tRNA fragments observed in shuttle RNA abundant transcript matching a region located 3 kB could represent tRNA halves, which are produced by a upstream of the Tia1 gene, coding for T cell intracellular single cleavage event in the anticodon loop that cuts the 0 0 antigen 1, which is involved in apoptosis and mRNA mature tRNA into one 5 and one 3 halve. It has been sorting into stress granules. This non-annotated transcript shown that these halves are often not present at equal locates to a highly conserved region and might be classi- quantities, differ in functions, such as the induction of fied as PROMPT (28). The function of PROMPs is largely stress granule assembly (62), and can be recruited to unknown, but could involve positively or negatively distinct cytoplasmic structures (63). Our observation that influencing the expression of downstream located genes. different cleavage products of the same RNAs distributed Future studies on the large number of (non-)annotated unequally over cellular and shuttle RNA further intergenic elements enriched in shuttle RNA may strengthens the concept that cells select specific RNAs uncover additional gene regulatory sequences that are for extracellular release. We also observed that the transferred between cells. Other issues that need to be ad- sequence coverage on particular tRNA genes was different dressed in the future include the deep sequencing of the in shuttle RNA compared with cellular RNA. This could larger, 70–200 nt fraction of small shuttle RNA and indicate that specific tRNA cleavage processes are determining the cellular origin (DC or T cell) of the involved in the generation of tRFs for dissemination to observed RNA species. The RNA in DC- and T the extracellular space. cell-derived vesicles could for example be separately analyzed after absorption of vesicles released in DC-T Infrastructural RNAs cell co-cultures onto beads coated with cell-type specific We observed a remarkable enrichment of sequences antibodies. matching SRP-RNA and the less studied infrastructural Since the relative amount of small cellular RNA se- RNAs vault- and Y-RNA in shuttle RNA. Similar to quences that map to 18S and 28S rRNA is low, we tRNAs, all of these structural RNAs are transcribed by expect that experimentally induced RNA degradation in Polymerase III and function in association with cytoplas- our studies is limited. Although some of the abundant mic proteins. The full-length forms of these RNAs are RNA species detected in shuttle RNA are cleavage longer than 70 nt, and only fragments were detected by products derived from mature non-coding RNAs, it is sequencing. Small vRNA fragments (svRNA) covering not likely that shuttle RNA represents non-selective 0 0 the 5 and 3 end of the vRNA have been detected in disposal of RNAs that have been degraded inside cells. human cells (32), and we detected similar fragments in Non-selective disposal of degraded RNAs would lead to cellular RNA derived from mouse DC-T cell co-cultures. a much more similar distribution of the degradation Interestingly, in shuttle RNA we detected coverage on a products over cells and vesicles than indicated by our different stem-loop structure. Future studies should reveal deep sequencing results. In fact, we even observed a whether this specific fragment of vRNA can also be pro- highly unequal distribution of different degradation cessed by Dicer and whether it can function in regulation products of the same RNAs over cellular and shuttle of gene expression. Indications based on qPCR analysis RNA. Furthermore, evidence accumulates that (partly) suggest that not only fragments, but also full-length forms degraded RNA fragments, such as tRFs, can also act as of SRP- and Y-RNA were present in shuttle RNA, more regulatory RNAs influencing gene expression. specifically, in low-density vesicles released by the cells. Uncontrolled release of these fragments would therefore The relative high amounts of full-length SRP- and impose a risk on disturbing cellular homeostasis. Y-RNA (Figure 6B) in cell-derived vesicles may explain By classification and quantification of the deep the higher enrichment of SRP- and Y-RNA observed by sequencing data, we gained important information on qPCR in comparison with fragment sequencing. Y- and the type of RNAs selected by immune cells for extracellu- SRP-RNAs belong to a small group of 5–6 host RNAs lar release. This study revealed that shuttle RNA contains that are also selectively incorporated together with viral a wealth of different non-coding small RNAs. Many of 9284 Nucleic Acids Research, 2012, Vol. 40, No. 18 11. Nolte-’t Hoen,E.N., Buschow,S.I., Anderton,S.M., Stoorvogel,W. the highly abundant small non-coding transcripts present and Wauben,M.H. (2009) Activated T cells recruit exosomes in shuttle RNA are evolutionary well conserved and have secreted by dendritic cells via LFA-1. Blood, 113, 1977–1981. previously been associated to gene regulatory functions. 12. Harding,C., Heuser,J. and Stahl,P. (1983) Receptor-mediated These findings allude to a wider range of biological effects endocytosis of transferrin and recycling of the transferrin receptor that could be mediated by shuttle RNA than previously in rat reticulocytes. J. Cell Biol., 97, 329–339. 13. Johnstone,R.M., Adam,M., Hammond,J.R., Orr,L. and anticipated. Gene regulatory functions of shuttle RNA Turbide,C. 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Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions

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The Author(s) 2012. Published by Oxford University Press.
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Abstract

9272–9285 Nucleic Acids Research, 2012, Vol. 40, No. 18 Published online 19 July 2012 doi:10.1093/nar/gks658 Deep sequencing of RNA from immune cell-derived vesicles uncovers the selective incorporation of small non-coding RNA biotypes with potential regulatory functions 1, 2 1 Esther N. M. Nolte-’t Hoen *, Henk P. J. Buermans , Maaike Waasdorp , 1 1 2 Willem Stoorvogel , Marca H. M. Wauben and Peter A. C. ’t Hoen Department of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 2, 3584 CM Utrecht and Center for Human and Clinical Genetics, Leiden University Medical Center, Einthovenweg 20, 2300 RC Leiden, The Netherlands Received March 14, 2012; Revised June 13, 2012; Accepted June 14, 2012 INTRODUCTION ABSTRACT Nano-sized membrane vesicles represent a recently Cells release RNA-carrying vesicles and membrane- identified class of intercellular communication vehicles free RNA/protein complexes into the extracellular operating in many organisms (1–6). Such vesicles can milieu. Horizontal vesicle-mediated transfer of derive from multivesicular bodies (MVBs), which are such shuttle RNA between cells allows dissemin- late endosomal compartments containing multiple ation of genetically encoded messages, which may 50–100 nm sized intraluminal vesicles. Fusion of MVBs modify the function of target cells. Other studies with the plasma membrane causes the release of their used array analysis to establish the presence of intraluminal vesicles, which are then called exosomes (7). microRNAs and mRNA in cell-derived vesicles Alternatively, vesicles can be released by cells through direct shedding from the plasma membrane (1). Cells from many sources. Here, we used an unbiased can tightly regulate the release and molecular composition approach by deep sequencing of small RNA of these vesicles (8,9) and vesicle targeting depends on the released by immune cells. We found a large variety type and activation status of recipient cells (10,11). of small non-coding RNA species representing per- Despite their early description decades ago (12,13), the vasive transcripts or RNA cleavage products wide-spread occurrence of cell-derived vesicles and their overlapping with protein coding regions, repeat se- potential for tailor-made modulation of target cell quences or structural RNAs. Many of these RNAs behaviour has only been recognized during the last few were enriched relative to cellular RNA, indicating years. It is now clear that cell-derived vesicles are not that cells destine specific RNAs for extracellular only released by almost all cultured cell types, but are release. Among the most abundant small RNAs in also present in a wide range of body fluids (1). Since the molecular make-up and release of cell-derived vesicles is shuttle RNA were sequences derived from vault regulated by the producing cell, these vesicles are of RNA, Y-RNA and specific tRNAs. Many of the interest for disease-related biomarkers (14). Moreover, highly abundant small non-coding transcripts in extracellular vesicles may be used as therapeutic agents shuttle RNA are evolutionary well-conserved and (15,16). have previously been associated to gene regulatory Besides specific sets of lipids and proteins, cells can functions. These findings allude to a wider range of shuttle RNA into vesicles determined for release into the biological effects that could be mediated by shuttle extracellular space. This allows the conveyance of genet- RNA than previously expected. Moreover, the data ically encoded messages between cells (17). The first key present leads for unraveling how cells modify the publication on nucleic acids in cell-derived vesicles function of other cells via transfer of specific reported the presence of miRNA and mRNA in vesicles non-coding RNA species. derived from mast cells and the functional transfer of *To whom correspondence should be addressed. Tel: +31 30 2534336; Fax: +31 30 2535492; Email: [email protected] The Author(s) 2012. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Nucleic Acids Research, 2012, Vol. 40, No. 18 9273 RNA to vesicle-targeted cells (17). More recently, the MATERIALS AND METHODS luminal protein and RNA contents of cell-derived Cell culture vesicles were demonstrated to be delivered into the cyto- D1 cells were maintained in Iscove’s Modified Dulbecco’s plasm of recipient cells via fusion of vesicles with these medium supplemented with 2 mM Ultraglutamine cells (18). It is currently not known whether all vesicle (Biowhittaker), 10% heat inactivated fetal calf serum populations released by cells contain RNA. Although (FCS, Sigma-Aldrich), 100 IU/ml penicillin and 100 mg/ several studies indicated that the RNA composition of ml streptomycin (GIBCO), 50 mM b-mercaptoethanol cell-derived vesicles is different from the parental cell and 30% conditioned medium from GM-CSF producing (17,19–21), it is unknown how RNAs are selected for se- NIH 3T3 cells (R1). The p53-specific CD4 T-cell clone, cretion into the extracellular space. Furthermore, extracel- generated in a C57BL/6 p53–/– mouse (34) and provided lular RNA may also be associated with macromolecular by Prof C. Melief (Leiden University Medical Center, complexes that are not enclosed by a vesicle. We here Leiden, The Netherlands), was cultured as described pre- collectively refer to extracellular RNA as ‘shuttle RNA’. viously (8). For cognate DC/T cell co-culture conditions, Although the majority of circulating miRNAs in human DC were pre-loaded with 2.5 mM p53 peptide (amino acid plasma and serum were found to co-fractionate with 77–96) for 2 h and subsequently mixed in a 1:1 ratio with T proteins such as Argonaute2 (Ago2) and nucleophosmin, cells, and co-cultured for 20 h in medium containing over- which have been suggested to protect miRNAs from deg- night ultracentrifuged (100 000g) FCS and R1 conditioned radation in RNase-rich environments (22,23), it is cur- medium (to deplete bovine and R1 derived vesicles and rently unknown if vesicle-free RNAs can bind and large protein aggregates). All cultures were maintained modify the function of target cells. at 37 C, 5% CO . Cell death in DC and T cell cultures Next-Generation Sequencing (NGS) techniques have was less than 5% and cognate DC-T cell interactions did led to the discovery of large numbers of unexpected not lead to additional cell death in the indicated co-culture non-coding RNAs (24). These transcripts were found to period. Experiments were approved by the institutional overlap with exons, introns and intergenic regions (24–30). ethical animal committee at Utrecht University (Utrecht, Various non-coding transcripts found in close vicinity to The Netherlands). protein coding genes, such as promoter-associated long and short RNAs, transcription start site-associated Shuttle RNA isolation RNAs, and PROMoter uPstream Transcripts (PROMPTs) are suspected to act as regulatory elements 15  10 p53 peptide-pulsed DCs were co-cultured with 15  10 T cells in 25 ml culture medium as described to modulate gene activity (30). Interestingly, many small earlier. Supernatants were pooled to a total of 450 ml. non-coding RNA species have been found that could act This culture supernatant was subjected to differential cen- as regulatory RNAs similar to miRNAs. Fragments trifugation (35). In short, cells were removed by two se- derived from small nucleolar RNA (snoRNA), vault quential centrifugations at 200g for 10 min. Collected RNA (vRNA) and transfer RNA (tRNA), for example, supernatant was subsequently centrifuged two times at were shown to bind Argonaute (AGO) proteins and form 500g for 10 min, followed by 10 000g for 30 min. Vesicles RNA-induced silencing complexes (RISCs) capable of and protein/RNA complexes were finally pelleted by ultra- regulating expression of target mRNAs analogous to centrifugation at 100 000g for 65 min in SW28 and SW40 miRNA-containing RISCs (31–33). rotors (Beckman). When indicated, the 100 000g sedi- The analysis of shuttle RNA populations has almost mented material was further fractionated using gradient exclusively focused on miRNAs and mRNAs, most density centrifugation. To this end, the 100 000g sedi- likely due to the availability and ease of array hybridiza- mented material was mixed with 2.5 M sucrose, overlaid tion techniques to detect these RNAs. Which other small with a linear sucrose gradient (2.0–0.4 M sucrose in PBS) RNA biotypes were released by cells via vesicles or in and floated into the gradient by centrifugation in a SW40 protein complexes was unknown. We therefore aimed to tube (Beckman) for 16 h at 192 000g. The bottom two frac- comprehensively analyze small RNA species released by tions (1.26–1.28 g/ml, as measured by refractometry) and cells using NGS. Among the most well studied cell-derived fractions with densities of 1.12–1.18 g/ml were pooled, vesicles are those produced by dendritic cells (DCs). These diluted with PBS/0.1% BSA and centrifuged again at cells are the sentinels of the immune system and the inter- 100 000g for 65 min. Small RNA (<200 nt) was isolated action of DC with T cells is the key event in initiation of from 100 000g pellets and from co-cultured DC and T immune responses. We previously found that DC-T cell TM cells (15  106 each) using the mirVana miRNA interactions induce vesicle release by DC and that Isolation Kit (Ambion) according to the manufacturer’s DC-derived vesicles can be targeted to activated T cells procedure. The RNA integrity and size distributions were (8,11). The release and targeting of RNA-containing assessed using Agilent 2100 Bioanalyzer pico-RNA chips. vesicles by DC and T cells may play an important role in the communication between these cells and the Small RNA sequencing library generation ensuing immune response. Here, we extensively characterized small (<70 nt) shuttle RNA released Sequencing libraries were generated as described (36). In during DC-T cell interactions and compared this RNA short, small RNA fractions were ligated to adaptors using with small cellular RNA in order to investigate selectivity the SOLiD Small RNA Expression Kit (Ambion). in the extracellular release of RNA species. Libraries were pre-amplified using primers containing 9274 Nucleic Acids Research, 2012, Vol. 40, No. 18 sequences that make the SREK libraries compatible with 5p mmu-mir-191 forward: 5 -CGGAATCCCAAAAGCA the Illumina flow cell. DNA was denatured for 30 at 98 C GCTG 00  00  00 All forward miRNA primers were used in combination followed by 18 cycles with 30 at 98 C, 30 at 65 C, 30 at with the NCode universal reverse primer. 72 C and final extension for 5 at 72 C. Library fragments Y-RNA forward: 5 -GTGTTTACAACTAATTGATCA were separated on a native 6% gradient pre-cast PAGE CAACC gel (Novex, Invitrogen). The 100–150 bp size fractions Y-RNA reverse: NCode universal reverse primer containing inserts of 20–70 nt in length were excised, SRP-RNA forward: 5 -GGAGTTCTGGGCTGTAGT DNA was eluted from the gel, precipitated and dissolved GC in nuclease free water. DNA yield was quantified using an SRP-RNA reverse: 5 -ATCAGCACGGGAGTTTTG Agilent Bioanalyzer high sensitivity DNA chip. Small AC RNA inserts were single end sequenced for 35 cycles using standard Illumina protocols. Cycling conditions were as follows: 95 C for 10 min followed by 50 cycles of 95 C for 10 s, 57 C for 30 s and Small RNA sequence data analysis 72 C for 20 s. All PCR reactions were performed on the Bio-Rad iQ5 Multicolor Real-Time PCR Detection Adaptor trimming, genome alignment and small RNA an- System (Bio-Rad). Raw threshold cycle (Ct) values were notation were performed using the E-miR data analysis calculated using the iQ5 Optical System software v.2.0 pipeline as described (36). Sequence reads were aligned to using automatic baseline settings. Thresholds were set in the Mouse Mm9 reference genome with bowtie, allowing the linear phase of the amplification plots. for alignments to three loci with two mismatches, followed by a crossmapping correction as described by de Hoon et al. (37). For further annotation with coding and RESULTS repeat RNAs, reads with a minimum overlap of 1 nt were clustered into regions and regions containing a Sample preparation and sequencing total of more than five reads in shuttle and cellular Specific antigen-bearing DCs were co-cultured with RNA were annotated using custom perl scripts (38) with cognate T cells, leading to mutual activation of both cell 0 0 exon, 3 -UTR, 5 -UTR, intron and RNA biotype informa- types. Vesicles and large protein complexes released tion from Ensembl biomart (39), all in a strand-specific during these interactions were isolated from cell culture manner. Intersections with repeat regions were determined supernatant by differential centrifugation, with a final pel- using information from the repeat track in the UCSC leting step at 100 000g (35). Total RNA was isolated from genome browser (NCBI137/mm9) and the Galaxy inter- this fraction and we observed that the majority of this face, considering a minimum of 1 nt overlap between the shuttle RNA consisted of small RNAs (<200 nt), with sequenced and the repeat region. To determine reproduci- minor amounts of 18S and 28S rRNA (Figure 1A). bility of the sample preparation and sequencing protocols, Next, small (<200 nt) RNAs were isolated from the we have evaluated the Pearson’s correlation between the 100 000g sedimented material, and processed for analysis abundance of small RNAs in replicate experiments. A by NGS. To assess whether selective RNAs were released square root transformation on the number of counts per into the extracellular space, small RNA from cells that small RNA was applied to stabilize variance and reduce had released the shuttle RNA was analyzed in parallel. influence of high-abundant small RNAs on the Pearson’s Strand specific small RNA sequencing libraries were correlation coefficient (38). prepared and pre-amplified as described (36). Fragments with inserts between 15 and 70 nt were gel purified, after Quantitative real-time PCR which the first 35 bases of the library were read with a single read, with the Illumina platform. Fragments 70 nt Sequencing derived expression profiles were compared were not included to prevent the large number of tRNAs with qPCR measurements on small RNA fractions from (70 nt in size) present in the cellular RNA fraction to independent biological samples. cDNA was generated dominate the sequencing reaction, which would hamper using the Ncode miRNA First-Strand cDNA Synthesis quantitative comparison of the cellular and shuttle RNA kit (Invitrogen). An equivalent of 100 pg RNA was used fractions. Sequencing data were processed using a in the PCR reactions with 100 nM primers (Isogen) and modified version of the E-miR pipeline (36). The se- 4 ul SYBR Green Sensimix (Bioline) in an 8 ml reaction. quences were mapped to the mouse genome (NCBI137/ PCR amplification efficiencies were determined using mm9), using bowtie allowing for two mismatches and 10-fold dilution series of template DNA and were three alignments per sequence read, followed by a between 1.8 and 2.3 for all primer sets. Transcript crossmapping correction (37). specific forward primers for miR-29a, miR-155 and To validate the robustness of our sequencing results, we miR-191 were based on full-length miRbase sequences replicated the presented experiment with a slightly differ- (40): ent sample preparation method (including an extra RNA 3p mmu-mir-29a forward: 5 -TAGCACCATCTGAAAT fragmentation step). We found strong concordance when CGGTTA ranking the different RNAs based on their abundance in 5p mmu-mir-155 forward: 5 -GGGTTAATGCTAATTG the shuttle RNA. We calculated the correlation between TGATAGGG the (ranked) small shuttle RNA sequencing data of the Nucleic Acids Research, 2012, Vol. 40, No. 18 9275 [nt] Cellular RNA Shuttle RNA Mt_rRNA Mt_tRNA miRNA misc_RNA rRNA snRNA snoRNA cellular RNA shuttle RNA 7SK RNA SCARNA SRP-RNA Vault RNA Y-RNA Figure 1. Shuttle RNA mainly consists of small RNA species and contains relatively low amounts of miRNA. Cell culture supernatant of DC-T cell co-cultures was subjected to several differential centrifugation steps, followed by RNA isolation from the 100 000g sedimenting material (shuttle RNA). (A) Bioanalyzer electropherogram of total shuttle RNA derived from DC-T cell co-cultures. The composition of small (<200 nt) shuttle and cellular RNA was further analyzed by deep sequencing. (B) Sequence reads uniquely aligned to the genome were annotated to known non-coding RNA transcripts as annotated in Ensembl using the E-miR pipeline. Data represent the percentages of reads in the indicated categories of annotated transcripts in shuttle RNA (grey bars) versus cellular RNA (black bars) calculated relative to the total sum of reads for Ensembl non-coding RNA transcripts. (C) Distribution of shuttle and cellular RNA transcripts over the different RNA biotypes within the Ensembl-annotated miscellaneous RNA (misc_RNA) category. Data are represented as the percentages of annotated transcripts as in (B). replicates. A high correlation was observed between the RNAs over the different biotypes. We found that the replicates (Pearson’s correlation 0.97, P-value <2.2e-16; majority of sequences present in the cellular small RNA Supplementary Figure S1). population represented miRNAs (Figure 1B), which is in agreement with previous studies (36,41). In contrast, the percentage of miRNAs in shuttle RNA was very low. miRNAs are underrepresented in shuttle RNA compared Instead, this fraction contained a relatively higher percent- with cellular RNA age of small ribosomal RNA (rRNA) and RNA belonging An overview of the sequencing data analysis and results to the miscellaneous category (misc_RNA) (Figure 1B). can be found in Figure 2. We found that 8.3  10E Within this category of misc_RNA, we found that struc- cellular and 27.5  10E shuttle RNA sequences aligned tural SRP-RNA, vRNA and Y-RNA were most abundant to the mouse genome (Figure 2). Uniquely aligned in shuttle RNA (Figure 1C). sequence reads were annotated to known non-coding small RNA transcripts as annotated in Ensembl. The miRNA distribution in shuttle and Remarkably, the percentage of sequence reads mapping cellular RNA is different to this category of RNAs was much lower in shuttle RNA released from cells (0.5%) compared with cellular Since the initial discovery of miRNAs in cell-derived RNA (31%). Within this category of Ensembl-annotated vesicles (17), many research groups have studied the oc- non-coding small RNAs, we analyzed the distribution of currence of this type of regulatory RNAs in vesicles % of reads % of reads 9276 Nucleic Acids Research, 2012, Vol. 40, No. 18 Truncated sequences Cells: 9,887,963 Shuttle: 28,820,290 Aligned sequences Cells: 8,273,374 (83.7%) Shuttle: 27,500,560 (95.4%) Annotation EmiR pipeline Annotation exons, introns, and repeats Ensembl small Not Ensembl small Aligned and in regions with > 5 non-coding RNA non-coding RNA reads Cells: 2,568,765 (31.0%) Cells: 5,704,609 (69.0%) Cells: 4,880,148 (59.0%) Shuttle: 134,932 (0.49%) Shuttle: 27,365,628 (99.5%) Shuttle: 18,063,486 (65.7%) miRNA not miRNA Exons Introns Repeats Cells: 1,471,703 (57.3%) Cells: 1,097,062 (42.7%) Cells: 1,967,028 (40.3%) Cells: 512,776 (10.5%) Cells: 1,453,876 (29.8%) Shuttle:10,784 (7.8%) Shuttle:124,148 (92.2%) Shuttle: 1,255,824 (6.7%) Shuttle: 4,946,811 (27.4%) Shuttle: 3,502,030 (19.4%) Exons of protein Exons of non-protein coding regions coding regions Cells: 967,229 (49.2%) Cells: 999,799 (50.8%) Shuttle: 1,060,780 (84.5%) Shuttle: 195,044 (15.5%) Figure 2. Overview of the sequencing data analysis and results. Indicated are the number of reads in each analysis step for small cellular versus shuttle RNA and the relative amount of reads calculated as a percentage of the reads detected in the previous steps. derived from various cell types (as listed in the ExoCarta process, but that cells actively sort selective miRNAs for database (42)). Although miRNAs formed only a small extracellular destination. minority within the total pool of shuttle RNA sequences Shuttle and cellular RNA contain different sets of (Figure 1B), we examined whether a specific set of transcripts miRNAs was released during DC-T cell interactions. The miRNAs occurring in cells of the DC-T cell co-culture Since the shuttle RNA population contained only 0.5% and shuttle RNA released by these cells were ranked based Ensembl-annotated small non-coding RNA transcripts, on the E-miR data and the 10 most prevalent miRNAs in we next aimed to define which other types of transcripts each population were compared (Table 1). miRNAs were were present in this population. By comparison with 0 0 named according to the 5 (5p) or 3 (3p) arm of the genomic locations obtained from Ensembl Biomart (39), hairpin where they originated from (36). Certain we found that 26.1% of the shuttle RNA reads (against miRNAs, such as miR-29a and miR-31, were abundant 49.9% in cellular small RNA) mapped to introns and in both cellular and shuttle RNA. However, several highly exons (Figure 2). Cellular small RNAs showed a higher abundant cellular miRNAs, such as miR92a-1 and let-7b, frequency of exonic localization (including a large number occurred only in very low abundance in shuttle RNA. of exons coding for miRNAs). However, the relative Conversely, the highly abundant shuttle miRNAs amount of exonic sequences mapping to protein coding miR-223, miR-142 and miR-93 were less abundant in regions was higher in shuttle RNA. In addition, shuttle cellular RNA. These data indicate that dissemination of RNA contained relatively more intronic sequences miRNAs into the extracellular space is not a random (Figure 2). Data have recently accumulated demonstrating Nucleic Acids Research, 2012, Vol. 40, No. 18 9277 Table 1. Enriched miRNAs in cellular and shuttle RNA that protein-coding genes can also produce a complex set of non-coding RNAs (43), including transcripts miRNA miRNA arm Rank Rank in originating from introns and from the untranslated in cells shuttle RNA regions (UTRs) (30,43). Interestingly, we observed that Abundant cellular miRNAs the percentage of reads in protein coding loci that 0 0 miR-155 5p 1 29 aligned to 3 - and 5 -UTRs was much higher in shuttle miR-29a 3p 2 1 RNA compared with cellular RNA (Figure 3A). Most miR-92a-1 3p 3 860 of these transcripts had the same directionality as the miR-31 5p 4 5 miR-15b 5p 5 46 coding mRNA. Although their exact function is miR-744 5p 6 47 unknown, these UTR-derived small RNAs have been sug- miR-let-7b 5p 7 119 gested to play a regulatory role in the attenuation or regu- miR-191 5p 8 4 lation of translation (44). miR-24-2 3p 9 10 miR-24-1 3p 10 12 Further analysis of the exonic sequences in shuttle and Abundant shuttle miRNAs cellular RNA revealed that the large majority (84%) of miR-29a 3p 2 1 exonic reads in shuttle RNA annotated to protein miR-21 5p 15 2 coding transcripts (Figure 3B), preferentially locating in miR-223 3p 41 3 miR-191 5p 8 4 the UTR regions of those transcripts (Figure 3A). Also the miR-31 5p 4 5 relative abundance of the small non-coding vRNA, miR-142 5p 47 6 Y-RNA and SRP-RNA was higher in shuttle RNA miR-93 5p 48 7 compared with cellular RNA. In contrast, lincRNA and miR-103-1 3p 18 8 miR-103-2 3p 14 9 miRNA are less abundant in shuttle RNA compared with miR-24-2 3p 9 10 cellular RNA (Figure 3B). Taken together, these results suggest that shuttle RNA is enriched in non-coding RNAs miRNAs were ranked according to the frequency of their occurrence other than miRNAs and lincRNAs and that many of these (number of reads). Indicated are the 10 most prevalent miRNAs in cells or shuttle RNA and the corresponding ranking in the other sample. are still to be annotated. Cellular RNA Shuttle RNA sense sense antisense antisense 15 15 3'-UTR 5'-UTR 3'-UTR 5'-UTR cellular RNA 45 shuttle RNA Figure 3. Exonic hits in shuttle RNA preferentially locate to UTR regions. (A) Exonic hits were analyzed for overlap with UTR regions. Indicated is the percentage of reads in shuttle RNA (left) and cellular RNA (right) mapping to the sense or antisense strand of UTR-regions, calculated relative to the total number of reads in Ensembl annotated exons. (B) The identity of exonic hits in shuttle (black) and cellular (grey) RNA. Indicated are the percentages of reads in the different RNA biotypes calculated relative to the total number of exonic reads. lincRNA snoRNA miRNA processed transcript protein coding pseudogene rRNA snRNA snoRNA Vault Y R A - N SRP-RNA 7SK -RNA % of exon reads % of exonic reads in prot coding regions 9278 Nucleic Acids Research, 2012, Vol. 40, No. 18 cellular RNA shuttle RNA cellular RNA shuttle RNA Figure 4. Different distribution of RNA repeat sequences in shuttle and cellular RNA. Abundance and classification of sequencing reads that match different types of RNA repeats. Indicated are the percentages of all reads (A) or reads in the top 1000 of most abundant regions (B) in the indicated repeat types, as detected in shuttle (black) and cellular (grey) RNA. The distribution of RNA repeat sequences is different in repeat sequences (Figure 4B). The most prevalent types of shuttle and cellular RNA repeats in shuttle RNA were tRNAs, LTRs, LINEs and simple repeats. LTRs, LINEs and simple repeats were Since the majority of small shuttle RNA transcripts did relatively much more abundant in shuttle RNA not correspond to Ensembl-annotated small non-coding compared with cellular RNA (Figure 4B). or protein-coding RNAs, we next investigated whether the as yet undefined transcripts corresponded to Specific tRNA fragments are abundantly and selectively repeat-derived RNAs. The relative abundance of repeat present in shuttle RNA RNAs in shuttle and cellular RNA was approximately the same (+/ 25%, Figure 4A). However, the distribu- To investigate in greater detail the most abundant se- tion over the type of repeat sequences in these two RNA quences in shuttle RNA, all unique regions were ranked populations was different. Shuttle RNA contained rela- by abundance (number of reads found per region) and the tively large numbers of Long Interspersed Elements RNA biotypes of the top 75 highest ranked hits were (LINEs), Long Terminal Repeats (LTRs) and simple analyzed. For comparison, a similar top 75 ranking was repeat sequences, whereas the majority of repeat se- made for the sequences found in cellular RNA (Table 2). quences in the cellular RNA consisted of tRNAs We observed that specific snoRNA and miRNAs were (Figure 4A). To further classify the most abundant among the most abundant sequences in the cellular repeat-associated RNA species, we next considered only small RNA fraction, but not in shuttle RNA. However, the top 1000 of most prominently present shuttle and as expected from previous data (Figure 4B), the shuttle cellular RNA. The differences in the distribution of the RNA was more abundant in different types of repeat se- most abundant shuttle and cellular RNA were much more quences. The high abundance of tRNA hits in both the pronounced. Almost 90% of the 1000 most abundant se- shuttle and cellular RNA fractions was remarkable, since quences in shuttle RNA (versus 33% in cells) consisted of we restricted our sequencing analysis to <70 nt RNAs. tRNA scRNA snRNA rRNA srpRNA SINE tRNA LINE scRNA LTR n A simple repeats s RN low complexity repeats rRNA non repeats srpRNA SINE LINE LTR simplerepeats low complexity repeats non repeats % of reads in regions top 1000 % of total reads Nucleic Acids Research, 2012, Vol. 40, No. 18 9279 Consequently, the sequence reads were not likely to rep- shuttle RNA, fragments of tRNA-Asp-GAY were only abundant in cellular RNA. We observed that the resent mature full-length tRNAs. We next explored majority of abundant tRNA hits in shuttle RNA whether these reads could represent tRNA fragments covered larger regions of about 40–50 nt (Table 3 and (tRFs), which have recently gained interest due to their Figure 5), caused by the presence of reads representing suspected regulatory nature (45). tRNA fragmentation is tRNA fragments (rather than full-length tRNAs) a specific process, with the composition, abundance and missing the first 5–15 nt of the mature tRNA. cleavage site varying per organism and cell type. Remarkably, at the same genomic locations, the Fragments of 18–22 nt, 30–35 nt and 50 nt locating at 0 0 coverage in cellular RNA was restricted to 30–35 nt frag- the 3 -or5 - end of mature tRNAs have previously been ments (Figure 5), indicating the presence of two different described (45–47). In cellular RNA, we observed frag- 0 0 fragments of the same tRNA, of which one is uniquely ments located at the 5 or 3 end of the mature tRNA present in shuttle RNA. (Table 3). However, the most abundant tRNA hits in shuttle RNA were all located at the 5 end of mature A specifically cleaved part of vRNA is abundantly present tRNAs. Differences were also observed in the type of in shuttle RNA tRNAs occurring in the top 75 most abundant hits of cellular and shuttle RNA; whereas fragments of Apart from the repeat and tRNA fragments, three other tRNA-Lys-AAA were abundant in both cellular and RNA biotypes were abundantly present in shuttle RNA (Table 2): (i) vRNA; (ii) SRP-RNA (7SL-RNA); and (iii) Y-RNA. All three RNAs are non-coding polymerase III RNA transcripts. Due to our <70 nt size restriction, we Table 2. Most abundant biotypes in cellular and shuttle RNA did not expect to find full-length transcripts of these three RNA biotype No. of reads No. of regions RNA biotypes. vRNAs are a family of RNAs found associated with the vault ribonucleoprotein complex Cellular RNA located in the cytoplasm (48). The function of these snoRNA 615 728 20 vault particles are largely unknown, but they are miRNA 599 709 20 tRNA repeat 586 583 27 thought to play a role in transportation of molecules, Unclassified 379 199 4 such as mRNA, from the nucleus to the cytoplasm and rRNA 107 855 2 in drug metabolism (e.g. in tumor cells). Mouse vRNA is Protein coding 103 877 1 141 nt long, but it was recently discovered that small vault vRNA 15 776 1 Shuttle RNA RNAs (svRNAs) can be generated from vault non-coding tRNA repeat 670 645 47 RNAs through a DICER-dependent and Simple repeat 484 945 6 DROSHA-independent mechanism (32). These svRNAs LINE 325 549 5 can downregulate expression of specific genes, similar to rRNA 120 797 5 miRNAs. Interestingly, we found that the predominant vRNA 45 546 1 Protein coding 26 492 4 coverage on the vRNA in exosomes was in only one of SRP-RNA 18 553 2 the internal stem loop structures (Figure 6A, B). In Y-RNA 5929 1 contrast, the coverage in cellular RNA was predominantly 0 0 at the 3 and 5 ends of vRNA, resembling the localization Regions were ranked by abundance (number of reads per region) and the top 75 highest ranked hits were categorized based on RNA biotype. of the described human svRNAs (32). These data indicate Indicated are the collective number of reads mapping to the different that a specifically cleaved part of the vRNA is preferen- biotypes and the number of different regions over which these reads tially shuttled into the extracellular space. were distributed. Table 3. Most abundant tRNA fragments in cellular and shuttle RNA tRNA fragment Location No. of reads No. of regions Region length (nt) Abundant cellular tRNA hits tRNA-Asp-GAY 3 -end 389 792 14 35 tRNA-Lys-AAG 5 -end 106 551 6 30–35 tRNA-Lys-AAA 5 -end 36 995 3 30 tRNA-Lys-AAG 3 -end 18 079 1 35 nt Abundant shuttle tRNA hits tRNA-Lys-AAA 5 -end 246 384 6 35 tRNA-Lys-AAG 5 -end 134 033 9 50 tRNA-Gly-GGA 5 -end 85 930 7 50 nt tRNA-Gly-GGY 5 -end 81 737 9 40 tRNA-Val-GTG 5 -end 44 160 5 50 tRNA-Glu-GAG 5 -end 27 900 5 40 tRNA-Val-GTA 5 -end 13 198 2 40–50 Hits in tRNAs in the top 75 highest ranked hits were annotated with anticodon, location of the region within the tRNA coding sequence (3 -or 5 -end), collective number of reads (accumulative for different copies of the same type of tRNA), the number of regions over which these reads were distributed, and the length of the regions. 9280 Nucleic Acids Research, 2012, Vol. 40, No. 18 Shuttle RNA Cellular RNA 5’ 3’ 3’ 5’ Coverage shuttle and cellular RNA Extra coverage shuttle RNA anticodon Figure 5. Different coverage of tRNA genes in shuttle and cellular RNA. (A) Screen shots were taken from the UCSC genome browser (http:// genome.ucsc.edu/) and show examples of tRNAs. Custom tracks were added to show sequence coverage in shuttle and cellular RNA. An example is shown of a tRNA gene (tRNA-Lys-AAG) on which differential coverage was detected in shuttle RNA (top) and cellular RNA (bottom). The y-axis represents the coverage at each genomic position. (B) tRNAscan-SE-predicted (http://lowelab.ucsc.edu/tRNAscan-SE) secondary structure of the mouse tRNA gene (tRNA-Lys-AAG) presented in (A). Indicated are the regions with coverage in shuttle and cellular RNA (dotted line), the additional coverage in shuttle RNA (black line), and the anticodon (circle). and RNAs (50). We found a 28 nt Y-RNA fragment and SRP- and Y-RNA are highly enriched in cell-derived 26–50 nt SRP-RNA covering regions in shuttle RNA. The vesicles abundance of SRP- and Y-RNA in material released by SRP-RNA (bound to the signal recognition particle) and cells into the extracellular space is remarkable, since these Y-RNA (bound to the Ro ribonucleoprotein complex RNAs belong to a small group of host RNAs that is also (49)) both function in intracellular transport of proteins selectively incorporated together with viral genomes into Nucleic Acids Research, 2012, Vol. 40, No. 18 9281 Main coverage shuttle RNA A B Main coverage cellular RNA Shuttle RNA Cellular RNA 5’ 3’ Figure 6. Different coverage of vRNA in shuttle and cellular RNA. (A) Screenshot from the UCSC genome browser showing the sequence coverage on the vRNA gene in shuttle RNA (top) and cellular RNA (bottom). The y-axis represents the coverage at each genomic position. (B) MFOLD-predicted (http://mfold.rna.albany.edu/) secondary structure of mouse vRNA at 21 C. Indicated are the regions with predominant coverage in shuttle RNA (black line) and cellular RNA (dotted lines). the capsids of several different viruses (51,52). To establish DISCUSSION a link between viruses and cell-derived vesicles regarding The complexity and diversity of the pools of extracellular incorporation of these RNAs, we investigated whether RNA in cell-derived vesicles and vesicle-free complexes is full-length SRP- and Y-RNA released in our DC-T cell daunting. Many research groups have focused on co-cultures were indeed associated to membrane vesicles. analyzing miRNAs in material released from a broad Hereto, the 100 000g material that was sedimented from range of cell types. However, the size distribution of the culture supernatant was further fractionated by shuttle RNA, as shown here and by others (53,54), gradient density ultracentrifugation. This allowed separ- extends well beyond the 20–23 nt size of miRNAs and is ation of RNA associated to large protein complexes, mostly in the range of 20 to 200 nt. This indicates that which are retained in the bottom of the gradient, and also other small RNA species are released by cells into the RNA enclosed in membrane vesicles, which float to low- extracellular space. By deep sequencing of the <70 nt buoyant densities (1.12–1.18 g/ml (35); Figure 7A). fraction of this shuttle RNA, we found a large variety of Subsequently, the presence of SRP- and Y-RNA in the small non-coding RNA species representing pervasive different density fractions was analyzed by RT-qPCR in transcripts or cleavage products overlapping with independent experiments. For comparison, the presence of protein coding regions, repeat sequences, or structural these RNAs was analyzed in the cellular <200 nt RNA RNAs. We extended this analysis with detailed evaluation fraction obtained from the DC-T cell co-culture from of the sequence coverage of several of the most abundant which the shuttle RNA was derived. Invariant endogenous RNA biotypes in shuttle RNA. This led to the discovery controls (reference genes) for qPCR analysis of that cellular and shuttle RNA can contain different small vesicle-enclosed RNA are unknown. As an alternative, RNAs derived from the same non-coding RNA, as was all samples were normalized to the total input quantity the case for tRNA and vRNA. Although no conclusions of RNA. Various miRNAs, which were relatively can be drawn regarding the absolute concentration of dif- abundant in cellular and/or shuttle RNA (Table 1), were ferent RNA species, the data allow comparison of the analyzed in parallel as a control. We observed that full- relative amounts of RNA types in the pools of cellular length SRP- and Y-RNA indeed associated to low-density and shuttle RNA. The unequal distribution of the cell-derived vesicles and not to high-density complexes. detected RNA species over cellular and shuttle RNA, Moreover, these RNAs were highly enriched in vesicles combined with increasing evidence for their role in gene compared with cells (Figure 7B). In contrast, all tested regulation strongly suggest that cells specifically release miRNAs were relatively more abundant in cells these RNAs to modify the function of target cells. compared with cell-derived vesicles, confirming the sequencing data (Figure 7C). Conclusively, SRP- and miRNA Y-RNA are highly abundant in cell-derived vesicles, similar to the enrichment of these host RNAs in virions. From both the sequencing and RT-qPCR data it became These findings may point to an evolutionary conserved clear that miRNAs were underrepresented in shuttle RNA mechanism by which cellular RNA and viral genomes compared with cellular RNA (Figures 1B and 7C). are selected and/or stabilized in membrane vesicles with However, the sequencing data revealed that the miRNA extracellular destination or by which transferred RNA can composition in shuttle RNA is not a mere reflection of the function in cells targeted by these vesicles. cellular miRNA (Table 1), indicating that a specific set of 9282 Nucleic Acids Research, 2012, Vol. 40, No. 18 Low density 1.12-1.18 g/ml High density 1.26-1.28 g/ml Y-RNA SRP-RNA 50 50 10 10 cells low dens high dens cells low dens high dens miR-191 miR-29a miR-155 1.2 1.2 1.2 1 1 0.8 0.8 0.8 0.6 0.6 0.6 0.4 0.4 0.4 0.2 0.2 0.2 0 0 0 cells low dens high dens cells low dens high dens cells low dens high dens Figure 7. Culture supernatant of DC-T cell co-cultures was subjected to consecutive differential centrifugation steps. The 100 000g pelleted material was loaded at the bottom of a sucrose gradient, after which low-density vesicles were floated to equilibrium density by ultracentrifugation. Low-density fractions (1.12–1.18 g/ml) and high-density (1.26–1.28 g/ml) bottom fractions of the gradient were collected (A). Small RNA (<200 nt) was isolated from the 100 000g pelleted material present in these fractions. (B, C) RT-qPCR analysis of Y- and SRP-RNA (B) and different miRNAs (C) in small cellular RNA (cells), 100 000g pelleted vesicles floating to low-density fractions (low dens) and 100 000g pelleted non-floating high-density material (high dens). All samples were normalized to input quantity of RNA. Relative expression levels were calculated based on the Ct values. Indicated is the fold increase ± s.d. of PCR product in floating or non-floating material relative to cells (set to 1). miRNAs is selected for extracellular release. Evidence RNA repeats exists that miRNAs associated to cell-derived vesicles are We found that repeat sequences were highly enriched in functional upon transfer to target cells (17,18). Although shuttle RNA compared with cells and that especially the the relative amounts of released miRNAs detected here LINE, LTR and simple repeats were more abundant in may appear low, significant effects on target cell function- shuttle RNA. Due to the size restriction of RNAs ing could be imposed due to efficient and specific cellular selected for sequencing, the detected transcripts do not targeting of vesicles and the capacity of miRNAs to cause represent full-length LINE or LTR. Small RNAs derived large-scale and long-term modulation of gene expression. from these repeats (repeat-associated small interference Interestingly, 5 out of the 10 most abundant shuttle RNA) have previously been shown to originate from miRNAs have validated target genes that play important various scattered regions within repeats such as SINE, roles in immune regulation. miR-93, for example, targets LINE and LTR (57,58). Interestingly, retrotransposon Stat3, which is involved in regulation of T cell responses RNA was recently found enriched in microvesicles (55), and miR-223 targets Mef2c, which is necessary for derived from tumor cells (59). It is currently unknown the transcriptional activation of interleukins during per- whether this retrotransposon RNA can be transferred to ipheral T cell activation (56). The sequencing and qPCR other cells and whether it can insert into the target studies described here have been performed on total cell genome. However, the dissemination and active tar- shuttle RNA obtained from DC-T cell co-culture super- geting of retrotransposable elements or fragments natant. Future studies will reveal which miRNAs (and thereof to other cells may be an effective strategy of other small non-coding RNAs) are released by DC or by T cells and which target cells can be modified by this cells to modify genes and regulate gene expression in shuttle RNA. other cells. Fold increase Fold increase Nucleic Acids Research, 2012, Vol. 40, No. 18 9283 A large number of sequences detected in shuttle RNA genomes into capsids of several different viruses (51,52). It mapped to tRNA loci. Full-length tRNAs were, however, is suggested that these host RNAs are encapsidated to excluded from analysis by size selection of RNAs smaller enhance virus assembly, virion stability and/or viral infect- than 70 nt. Although we cannot exclude low-level contam- ivity. Many enveloped viruses exploit the existing routes of ination with full-length tRNA, most tRNA hits will rep- membrane traffic to leave the host cell. A relationship between endosomal and/or plasma membrane routing of resent tRNA fragments (tRFs). Many recent publications viruses and cell-derived vesicles can therefore be envi- have demonstrated that tRFs are not merely degradation sioned (64). We hypothesize that these RNAs could play products but are specific cleavage products with versatile a role in the specific sorting of regulatory RNAs into functions, such as the inhibition of translation and cell-derived vesicles. Alternatively, these RNAs could sta- guidance of other RNAs (60). We here demonstrated bilize the RNA content of cell-derived vesicles or guide that the tRF compositions in cellular and shuttle RNA regulatory RNAs for efficient functioning upon release were different, indicating selectivity in released tRFs. into the cytoplasm of target cells. Earlier evidence that tRFs can be released from cells The abovementioned RNA biotypes make up only and transported to distant cells comes from the field of around 50% of the total transcripts present in shuttle plant physiology, where specific tRFs were identified in RNA. We found that many of the other transcripts the phloem sap of several different plants (61). The 5 located to intergenic regions. One example is a highly end located tRNA fragments observed in shuttle RNA abundant transcript matching a region located 3 kB could represent tRNA halves, which are produced by a upstream of the Tia1 gene, coding for T cell intracellular single cleavage event in the anticodon loop that cuts the 0 0 antigen 1, which is involved in apoptosis and mRNA mature tRNA into one 5 and one 3 halve. It has been sorting into stress granules. This non-annotated transcript shown that these halves are often not present at equal locates to a highly conserved region and might be classi- quantities, differ in functions, such as the induction of fied as PROMPT (28). The function of PROMPs is largely stress granule assembly (62), and can be recruited to unknown, but could involve positively or negatively distinct cytoplasmic structures (63). Our observation that influencing the expression of downstream located genes. different cleavage products of the same RNAs distributed Future studies on the large number of (non-)annotated unequally over cellular and shuttle RNA further intergenic elements enriched in shuttle RNA may strengthens the concept that cells select specific RNAs uncover additional gene regulatory sequences that are for extracellular release. We also observed that the transferred between cells. Other issues that need to be ad- sequence coverage on particular tRNA genes was different dressed in the future include the deep sequencing of the in shuttle RNA compared with cellular RNA. This could larger, 70–200 nt fraction of small shuttle RNA and indicate that specific tRNA cleavage processes are determining the cellular origin (DC or T cell) of the involved in the generation of tRFs for dissemination to observed RNA species. The RNA in DC- and T the extracellular space. cell-derived vesicles could for example be separately analyzed after absorption of vesicles released in DC-T Infrastructural RNAs cell co-cultures onto beads coated with cell-type specific We observed a remarkable enrichment of sequences antibodies. matching SRP-RNA and the less studied infrastructural Since the relative amount of small cellular RNA se- RNAs vault- and Y-RNA in shuttle RNA. Similar to quences that map to 18S and 28S rRNA is low, we tRNAs, all of these structural RNAs are transcribed by expect that experimentally induced RNA degradation in Polymerase III and function in association with cytoplas- our studies is limited. Although some of the abundant mic proteins. The full-length forms of these RNAs are RNA species detected in shuttle RNA are cleavage longer than 70 nt, and only fragments were detected by products derived from mature non-coding RNAs, it is sequencing. Small vRNA fragments (svRNA) covering not likely that shuttle RNA represents non-selective 0 0 the 5 and 3 end of the vRNA have been detected in disposal of RNAs that have been degraded inside cells. human cells (32), and we detected similar fragments in Non-selective disposal of degraded RNAs would lead to cellular RNA derived from mouse DC-T cell co-cultures. a much more similar distribution of the degradation Interestingly, in shuttle RNA we detected coverage on a products over cells and vesicles than indicated by our different stem-loop structure. Future studies should reveal deep sequencing results. In fact, we even observed a whether this specific fragment of vRNA can also be pro- highly unequal distribution of different degradation cessed by Dicer and whether it can function in regulation products of the same RNAs over cellular and shuttle of gene expression. Indications based on qPCR analysis RNA. Furthermore, evidence accumulates that (partly) suggest that not only fragments, but also full-length forms degraded RNA fragments, such as tRFs, can also act as of SRP- and Y-RNA were present in shuttle RNA, more regulatory RNAs influencing gene expression. specifically, in low-density vesicles released by the cells. Uncontrolled release of these fragments would therefore The relative high amounts of full-length SRP- and impose a risk on disturbing cellular homeostasis. Y-RNA (Figure 6B) in cell-derived vesicles may explain By classification and quantification of the deep the higher enrichment of SRP- and Y-RNA observed by sequencing data, we gained important information on qPCR in comparison with fragment sequencing. Y- and the type of RNAs selected by immune cells for extracellu- SRP-RNAs belong to a small group of 5–6 host RNAs lar release. This study revealed that shuttle RNA contains that are also selectively incorporated together with viral a wealth of different non-coding small RNAs. Many of 9284 Nucleic Acids Research, 2012, Vol. 40, No. 18 11. Nolte-’t Hoen,E.N., Buschow,S.I., Anderton,S.M., Stoorvogel,W. the highly abundant small non-coding transcripts present and Wauben,M.H. (2009) Activated T cells recruit exosomes in shuttle RNA are evolutionary well conserved and have secreted by dendritic cells via LFA-1. Blood, 113, 1977–1981. previously been associated to gene regulatory functions. 12. Harding,C., Heuser,J. and Stahl,P. (1983) Receptor-mediated These findings allude to a wider range of biological effects endocytosis of transferrin and recycling of the transferrin receptor that could be mediated by shuttle RNA than previously in rat reticulocytes. J. Cell Biol., 97, 329–339. 13. Johnstone,R.M., Adam,M., Hammond,J.R., Orr,L. and anticipated. Gene regulatory functions of shuttle RNA Turbide,C. 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Nucleic Acids Research – Oxford University Press

Published: Oct 19, 2012

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