Distribution of ncRNAs expression across hypothalamic-pituitary-gonadal axis in Capra hircus

Distribution of ncRNAs expression across hypothalamic-pituitary-gonadal axis in Capra hircus Background: Molecular regulation of the hypothalamic-pituitary-gonadal (HPG) axis plays an essential role in the fine tuning of seasonal estrus in Capra hircus. Noncoding RNAs (ncRNAs) are emerging as key regulators in sexual development and mammalian reproduction. In order to identify ncRNAs and to assess their expression patterns, along the HPG axis, we sequenced ncRNA libraries from hypothalamus, pituitary and ovary of three goats. Results: Among the medium length noncoding RNAs (mncRNAs) identified, small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs) were found to be more abundant in ovary and hypothalamus, respectively. The observed GC content was representative for different classes of ncRNAs, allowing the identification of a tRNA-derived RNA fragments (tRFs) subclass, which had a peak distribution around 32–38% GC content in the hypothalamus. Differences observed among organs confirmed the specificity of microRNA (miRNA) profiles for each organ system. Conclusions: Data on ncRNAs in organs constituting the HPG axis will contribute to understanding their role in the physiological regulation of reproduction in goats. Keywords: miRNA, HPG, Goat, Small-RNA, Reproduction Background Noncoding RNAs (ncRNAs) are involved in a remark- The hypothalamic-pituitary-gonadal (HPG) axis regu- able variety of biological functions. These RNAs are lates reproduction in mammals from fetal development, divided into several families based on their size and through puberty to sexual maturity [1]. The coordin- biogenesis pathways, and act as part of RNA-protein com- ation of peripheral organs with the central nervous plexes in regulating gene expression [4]. Regulatory system ensures that animal physiology is aligned with ncRNAs can be placed in three major classes based on the external environment to optimize reproductive transcript size: small (sncRNAs), medium (mncRNAs) and success [2]. The goat oestrous cycle is accompanied by long noncoding RNAs (lncRNAs) [5]. These different hormonal changes along the HPG axis, that orchestrate ncRNAs are further classified based on sequence or struc- morphological and physiological changes in the ovaries ture conservation, subcellular localization and function, leading to ovulation and preparation of the reproductive association with annotated protein-coding genes and tract for oocyte maturation, sperm transport, fertilization, other DNA elements of known function [6]. Various and embryo implantation [3]. bioinformatic tools are available that use either sequence motifs or structural parameters to detect novel ncRNAs [7]fromsequence data. * Correspondence: stella@ibba.cnr.it Various classes of ncRNAs have roles in promoting Emanuele Capra and Barbara Lazzari contributed equally to this work. the mammalian sexual phenotype [8]. Antisense long Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle non coding RNAs (lncRNAs) may affect the expression Ricerche, Lodi, Italy Parco Tecnologico Padano, Lodi, Italy and function of genes regulating sex determination and Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Capra et al. BMC Genomics (2018) 19:417 Page 2 of 9 gonad development, such as the forkhead box L2 RNA isolation (FOXL2)[9]. The U17 short nucleolar RNA (snoRNA) Total RNA was isolated from each sample using Trizol has been shown to regulate a small non-coding RNA (Invitrogen, Carlsbad, CA) and purified by the NucleoSpin® produced from the Snhg3 introns which influence cellu- miRNA kit (Macherey-Nagel, Germany), following the lar cholesterol trafficking in the ovary, and thus may play protocol recommended by the manufacturer to prepare a role in regulating steroid hormone production and small and large RNA in one fraction (total RNA). RNA postnatal gonadal maturation [10]. The role of miRNAs concentration and quality was determined using an Agilent in modulating the HPG axis is well documented. For 2100 Bioanalyzer (Santa Clara, CA). The isolated RNA was example, in the hypothalamus Lin28/let-7 expression stored at − 80 °C. patterns are associated with the onset of puberty [11]. Lin28a and Lin28b expression decreases during the on- Library preparation and sequencing set of puberty transition and Lin28b expression in the Small noncoding RNA (sncRNA) libraries were gener- ovary may be affected by environmental cues to delay ated using the TruSeq Small RNA Library Preparation puberty and/or follicular development [12]. MiR-361-3p kit according to manufacturer’s instructions (Illumina). is involved in regulating FSH secretion in a pig pituitary The libraries were then pooled (3 goats for 3 organs) cell model [13]. Examination of genome-wide miRNA and purified on a Pippin Prep system (Sage Science, expression in goats suggested that miR-424-5p and miR- MA, USA) to recover two fractions of 125 to 167 nt 29a regulate muscle development [14] and that miRNAs (fraction 1, Illumina adapters included), containing play an important role in endometrial receptivity [15]. It mature miRNAs, and of 168 to 300 nt (fraction 2, was also observed that miRNA differ in their expression Illumina adapters included), containing other ncRNAs, in the three stages of hair follicle cycles in cashmere respectively. The quality and yield after sample prepar- goats [16], as well as in the ovary between pregnant and ation was measured with an Agilent 2200 Tape Station, non-pregnant individuals, with 294 miRNA upregulated High Sensitivity D1000 (Santa Clara, CA). Libraries were and 113 downregulated in pregnant goats [17]. quantified by Real Time PCR with KAPA Library Quan- Although miRNAs and other ncRNAs have shown to tification Kits (Kapa Biosystems, Inc. MA, United play a role in the regulation of tissue development and States). Libraries from fraction 1 and fraction 2 were se- function, little is known about their tissue specification quenced on Miseq desktop sequencer (Illumina) with that to organ functionality along the reproductive axis. 50-base and 150-base single reads, respectively. In the present study, the ncRNA repertoires of hypo- thalamus, pituitary and ovary were investigated in Capra Bioinformatic analysis hircus by use of NGS. All sequences were quality checked with FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/) Methods and trimmed with Trimmomatic (minimum sequence Animals and tissue collection quality 30 and minimum sequence length 14) [18]. Three adult female Saanen goats, aged 43.3 ± 3.2 months (mean ± SD) and weighing 55.0 ± 2.3 kg (mean ± SD), ncRNA reared in the same group and on the same farm were ncRNA sequences from fraction 2 from hypothalamus, sacrificed at the end of their productive life. The private pituitary and ovary were collected and separately col- owner agreed to yield them to the present research in- lapsed in silico into three non-redundant datasets with stead of the slaughterhouse with full consciousness the Fastx-Toolkit collapser tool (http://hannonlab.cshl. about the purpose of the Project. edu/fastx_toolkit/). These datasets were compared by The experimental design was approved by the Animal BLASTn to the RNAcentral database sequences [19] and Ethic Committee of the University of Milan. Animals were hits having at least 90% coverage and 90% similarity to transported, anesthetized (Ketamine, 5 mg/kg/IV and the RNAcentral entries were assigned to the RNA class Diazepan 1 mg/kg/IV), sacrificed by receiving a single of the corresponding RNAcentral sequence. Statistical intravenous (IV) bolus injection of a 10 mL solution of analyses were performed with EdgeR [20] and the GLM embutramide, mebezonium iodide and tetracaine hydro- model was applied to identify sequences with differential chloride (Tanax) and organs were collected according to expression among the three datasets. Differential expres- the European Directive 2010/63/EU on the protection of sion analyses across the three organs were run with the animals used for scientific purposes. Samples of the hypo- Bioconductor edgeR package (GLM model, FDR < 0.01 thalamus, pituitary and ovaries were collected from each and LogFC> 1.5). goat. The samples were immediately frozen in liquid ni- GC content of goat ncRNA sequences and entries present trogen and ground to fine powder using mortar and pestle in the small human noncoding RNAs DASHR database and stored at − 80 °C until RNA extraction. (http://lisanwanglab.org/DASHR/smdb.php#tabData)[21], Capra et al. BMC Genomics (2018) 19:417 Page 3 of 9 were calculated with Geecee (http://www.bioinformatics.nl/ Results cgi-bin/emboss/geecee). Libraries preparation NcRNA libraries obtained from hypothalamus, pituitary miRNA data analysis and ovaries have three major size peaks, corresponding miRNA detection and discovery were carried out with to 149, 201 and 268 bp (Fig. 1a). To represent the variety Mirdeep2 on Illumina high quality trimmed sequences of small RNAs in goat, two small RNA fractions from from fraction 1. Capra hircus miRNA sequences available pooled libraries (3 tissues for 3 animals) were selected: i) at MirBase (http://www.mirbase.org/)wereusedtoidentify “fraction 1” containing miRNAs (about 20–30 nt in known miRNA in the trimmed sequences. Known miRNA length) and ii) “fraction 2” containing other ncRNAs from related species (sheep, cow and horse) available at (70–140 nt); Illumina adapters were about 120 bp in size MirBasewerealsousedbyMirdeep2tosupport theidenti- (Fig. 1b). Both fractions were sequenced and analyzed fication of novel goat miRNAs. The Mirdeep2 quantifier separately for their miRNAs and ncRNAs content. Data module was used to quantify expression and retrieve counts are available in the Sequence Reads Archive (SRA), Bio- for the known and novel miRNAs. Differential expression Project accession number, SRP136431. analyses across the three organs were run with the Biocon- ductor edgeR package (FDR < 0.01 and LogFC> 1.5). ncRNA analysis MiRNA cluster analysis was performed with the Genesis Miseq sequencing of fraction 2 (ncRNA 70-140 nt) re- software to identify and visualize patterns within the sulted in 22,309,383 total of raw reads, with an average datasets [22]. MiRNA target prediction was performed by of 2,447,141 reads per sample. FastQC analysis grouped Ingenuity Pathway Analysis (IPA, Ingenuity System, www. trimmed sequences depending on the nucleotide length ingenuity.com). Human homologous miRNAs were ana- and GC content (Additional file 2). In order to classify lyzed with. the sequenced Capra hyrcus ncRNAs according to the microRNA Target filter (IPA) to attribute (experimen- known ncRNA classes available in literature, homology tally observed) target genes. Gene ontology (GO) classifi- searches against the sequence dataset of the RNAcentral cation of miRNA target mRNA was performed according non-coding RNA sequence database [19] were carried to classical GO categories, using the Cytoscape plug-in out (Fig. 2). A high percentage of reads was assigned to ClueGO which integrates GO [23] and enhances small nucleolar RNA (snoRNA), ribosomal RNA (rRNA) biological interpretation of large lists of genes. , transfer RNA (tRNA), lncRNA and signal recognition particle (SRP RNA), while small nuclear (snRNA) and miRNA validationby real time PCR qRT-PCR precursor RNA, ribonuclease P and MRP RNA (Rnase P RNA samples isolated from each organ were retro- RNA and RNAse MRP RNA), miscellaneous RNA (mis- transcribed with miScript II RT Kit following manufac- cRNA), miRNA, antisense RNA (asRNA), guide RNA turer’s instructions (Qiagen, Inc., Valencia, CA USA). (gRNA) and vault RNA (vRNA) were less abundant. The Quantitative Real Time PCR (RT-PCR) was carried out ncRNA size and GC content distributions were consist- on cDNAs with 7900HT Fast Real-Time PCR System ent across all three organs. The majority of the ncRNAs (Applied Biosystems, Carlsbad, California, USA). were in the 60–90 nt size range. There was also a large Reactions were carried out in 10 μl volumes containing number of lnRNAs, snRNAs and Rnase P RNAs reads 1 M of each primer, 2 μl cDNA (see above), and 5 μl2× ranging from 115 to 140 nt long. The distribution of Power SYBR® Green PCR Master Mix (Applied Biosys- gRNAs showed two peaks, corresponding to 60-90 nt tems) according to manufacturer protocols. The primers and 142-151 nt. Different ncRNA classes were distrib- used for chi-miR-141, chi-miR-7, chi-miR-9-5p and chi- uted according to GC content: sequences with low GC miR-10a-5p quantification, were designed using miR- percentage were observed for snoRNA (peak at 32–38%) primer software [24], (Additional file 1). For miR-124a- , snRNA and precursor RNA (peak at 39–42%); a GC 1 quantification, the bta-mir-124a-1 miScript Primer content near 50% was observed for lncRNA (peak at Assay (Qiagen, Inc., Valencia, CA USA) was used. 45–49%) and rRNA, SRP RNA, miscRNA, miRNA, Normalization used the small nucleolar snoRNA as ref- antisense RNA, guide RNA and vault RNA (peak at erence, C/D Box 95 SNORD95 miScript Primer 50–54%); and a high GC content was observed for (Qiagen, Inc., Valencia, CA USA). Negative controls tRNA, RnaseP and Rnase MRP (peak at 60–66%). using water in place of samples were performed along- This highlights that in most cases ncRNA classes are side each reaction. Reactions were run using the cycling characterized by a defined GC content. parameters of 95 °C for 10 min, plus 40 cycles of 95 °C To validate this observation and further explore the for 15 s, and 60 °C for 1 min. Relative expression levels intrinsic properties of ncRNAs, the GC content of en- and significance for each treatment were calculated tries present in the human sncRNAs DASHR database separately using the 2-Ct method [25]. [19], including sequences belonging to different ncRNA Capra et al. BMC Genomics (2018) 19:417 Page 4 of 9 Fig. 1 Small RNA libraries preparation. A Agilent 2100 bioanalyzer profile of a small RNA library obtained from RNA extracted from Hypothalamus, Pituitary and Ovary. B Agilent Tape station profile of a small RNA library fraction obtained by size-selection with pippinprep: miRNA libraries (144 bp), ncRNA libraries (198 and 266 bp). In circle sRNA libraries isolated in fraction 1 and fraction 2: a) 144 bp, b) 198 bp and (c) 266 bp. Illumina adapters were120 bp long classes (rRNA, snoRNA, snRNA, tRNA), was calculated. hypothalamus. MiRNA precursors were under-represented The distribution was similar to that observed for in the pituitary (Fig. 2). Statistical analysis, based on ncRNAs in Capra hircus (Fig. 3). However, ncRNA GC 1549 ncRNAs expressed in three organs, revealed that distribution between goat and human presented some 8, 147 and 94 ncRNAs were differentially expressed be- differences, that could be probably related to the differ- tween pituitary, hypothalamus and ovary (FDR < 0.01) ent dataset used, repository and sequencing data for hu- (Additional file 3). The hypothalamus had a high pro- man and goat respectively. portion of tRNAs, whereas the ovary was enriched for The DASHR database contains the tRNA-derived snoRNAs (Additional file 4). RNA fragments (tRFs), which had a peak at 32–38% GC content. A similar GC content was observed for the miRNA analysis tRNA class in the hypothalamus that was probably asso- Miseq sequencing of fraction 1 (trimmed ncRNA of ciated with the presence of tRFs in this organ (compare about 20-30 nt in length) resulted in 12,592,015 total Fig. 2 and Fig. 3). Expression levels of other ncRNA clas- raw reads, with an average of 1,399,112 reads per sam- ses differed among organs, i.e. miscRNA, snoRNA and ple. The miRNA content in ncRNA libraries was ex- precursor RNA were more abundant in the ovary, plored by bioinformatic analysis of sequenced products lnRNA in the pituitary and tRNA and vRNA in the using the miRDeep2 software. 785 known and putative Capra et al. BMC Genomics (2018) 19:417 Page 5 of 9 Fig. 2 Distribution of different ncRNA classes in function of nucleotide length (nt) and GC content percentage (GC content) for the reads mapped against the RNAcentral database sequence collection. For each category the relative aboundance in each organis was reported (Y axis) miRNAs were identified and quantified in the tested pathway analysis revealed that many genes were involved samples. Among these miRNAs, 402 were already in fibroblast growth factor and epidermal growth factor known in Capra hircus (chi-miRNAs), 222 had hom- response in the pituitary. Pathways related to the ology with known miRNAs from other species and 161 regulation of macromolecule metabolic process, organ were predicted candidate novel miRNAs. After applying development, cellular and developmental processes were a stringent filtering (FDR < 0.01) for each target organ, prevalently targeted by the miRNA found upregulated in 87, 70, and 233 miRNA were identified that were differ- the hypothalamus and ovary (Additional file 6). entially expressed between pituitary, hypothalamus and Differential expression of specific miRNAs in each ovary, respectively. The differential expression was ob- organ was confirmed by qRT-PCR. MiR-141 and miR-7 tained by comparing the expression in one organ versus were highly expressed in the pituitary and miR-9 and the expression in the other two organs (Fig. 4). A list miR-124 highly expressed in the hypothalamus, whereas of organ-specific overexpressed miRNAs is given in miR-10a-5p had the highest level of expression in the Additional file 5. ovary (Additional file 7). Target genes of organ specific upregulated miRNAs found in this study were predicted, and related pathways Discussion identified. 6, 13 and 25 miRNA upregulated in pituitary, Goat mncRNA profiling of three organs according to hypothalamus and ovary targeted 54, 329 and 970 ex- GC content, showed five major peaks (GC content of perimental observed mRNA respectively. The canonical peaks: 32–38%, 39–42%, 45–49%, 50–54%, and 60–66%), Capra et al. BMC Genomics (2018) 19:417 Page 6 of 9 Fig. 3 a total GC content distribution and (b) defined range GC content distribution, calculated with the EMBOSS geecee software on ncRNA entries from the small human noncoding RNAs DASHR database and on ncRNAs (experimentally observed data) for all three organs hypothalamus (Hyp), pituitary (Pit) and ovary (Ov) together in Capra hircus. rRNA, snoRNA, snRNA, tRNA were present in both DASHR database and experimental dataset. tRF-RNA class was present only in the DASHR database. On the Y axis percentage of the relative aboundance of each category of ncRNAs was re- ported. The X axis reports the percentage of GC content for each group leading us to postulate that different mncRNAs have splicing through pre-mRNA secondary structures [32]. specific GC contents. Evaluation of distribution of GC GC content has also been found to influence the content for different classes of small human noncoding function of sncRNA. Short interfering RNA (siRNA) RNAs available in the DASHR database supports our re- GC-content correlates with RNA interference (RNAi) sults. Although GC content was one of the most useful efficiency [33]. GC-content of synonymous codons in features for separating ncRNAs from other genomic ele- coding sequences is proven to have an impact on amino ments [26, 27], we describe a deviation from random acid usage [34]. GC content for each mncRNA class. The relative abundance of the different mncRNA classes The CG content is an important feature that affects was similar in all three caprine organs: snoRNA, rRNA function and stability of RNA: CG rich mRNA is more and tRNA were the most represented, in agreement with efficiently translated, affecting protein products levels quantitative data on the expression landscape of small [28]. GC composition also influences the degradation human noncoding RNA from other tissues available in the rate of mRNAs [29] and lncRNAs [30] and affects DASHR database. stability of RNA secondary structure [31]. It has been In the current version of the database, the distribution suggested that GC content around splice sites affects of the various ncRNAs classes is different for different Capra et al. BMC Genomics (2018) 19:417 Page 7 of 9 Val Gly type of tRNA and tRNA derived fragments were observed to be specifically produced in a controlled fashion in rat brain exposed to ischemia [35]. The level of tRFs was observed to increase when tRF targets decreased with age in rat brain [36]. tRNA-derived small RNAs served as novel signaling molecules in the re- sponse to stress [37, 38]. The high level of tRF found in goat hypothalamus may be important for maintaining a correct epigenetic asset and regulating organ function. SnoRNAs regulate gene expression, playing a central role in ribosome biogenesis. However, many snoRNAs have not been ascribed a function, suggesting that they may have a different cell functionality [39]. Goat hypo- thalamus highly expresses SNORD109A, SNORD114 and SNORD116. Recently, an updated human snoR- NAome based on snoRNAs from RFAM-based predic- tions, generated by the GENCODE consortium, found SNORD116 family and SNORD109 to be specifically overexpressed in neurons [40]. Capra hircus ovary was enriched in many snoRNA. The relative overexpression of SNORD (58, 93, 19, 69, 101, 46, 58, 121A, 58, 19b, 24, 38, 12 and 106) in goat ovary, matched snoRNAs profiling between hypothal- amus and ovary from juvenile female sheep collected in Expression Atlas (http://www.ebi.ac.uk/gxa/home). On the contrary, three of the ovary overexpressed snoRNA in goat SNORD (18, 42 and 25) showed an opposite expression. MiRNAs are regulators of gene expression that exhibit tissue and developmental-specific patterns and contri- bute in maintaining tissue homeostasis [41, 42]. In the present study, several specific miRNAs were predomin- antly expressed in one particular organ. MiR-141, miR200a and miR-7 were expressed in the pituitary gland while miR-124, miR-128 and miR-9 were highly expressed in the hypothalamus. This has also been observed in rodents [41] and in humans [43]. In the present study high levels of expression of miR-10b, miR- 125b, miR-143, miR145, miR199b, miR21 and miR-99a were recorded in the ovary. A recent review identified that these miRNAs were highly expressed in mammalian Fig. 4 Hierarchical clustering obtained from normalized miRNA ovary [44]. count for each replicate (1, 2, 3) in the three organs: hypothalamus (Hyp), pituitary (Pit) and ovary (Ov). A subset of miRNAs showing the highest variance among organs is reported. Red indicates an increase in expression and green a decrease in expression relative to Conclusions the mean expression of 60 miRNAs In summary, this study described the goat (Capra hircus) ncRNA expression profiles in the three organs of the HPG axis. Comparison of these data with similar data tissues. We found that hypothalamus, pituitary and from other species, when it becomes available, will ovary ncRNAs content is specific for each of the goat or- provide insights into the role of different ncRNAs in the gans. The hypothalamus from goat was enriched for reproductive process. Finally, the ncRNA profiling may tRNAs and tRFs whereas ovary had an high level of serve as a reference for further studies investigating the snoRNAs. Intriguingly, goat hypothalamus expressed a peculiarities of goat reproductive physiology, including Gly(GCC) Val(AAC) high level of tRNA and tRNA . The same seasonality in both sexes. Capra et al. BMC Genomics (2018) 19:417 Page 8 of 9 Additional files Authors’ contributions GP, AS, JLW, PAM, BC2, PC conceived and designed the experiments. GP, PC, AS supervised the progress of the project. SF, EC, SC, BC1, AT, gathered Additional file 1: Primer list and sequences used for Real Time samples, prepared hypothalamus and ovary of the goats. BL, EC, AT, SF, PC validation experiment. (XLSX 9 kb) conducted the bioinformatics and statistical analysis. SF, EC conducted the Additional file 2: FastQC analysis result summary for ncRNA sequences experiments. EC, BL, SF, wrote the manuscript. All authors read and approved after trimming process. For each organ an example of the reads the final manuscript. distribution in function of sequence length and per sequence GC content was reported. (DOCX 217 kb) Ethics approval and consent to participate Animals were at the end of their productive career and the private owner Additional file 3: List of differentially expressed ncRNA (DE-ncRNA) agreed to yield them to the present research instead of the slaughterhouse (FDR < 0.01) for Pit (Pituitary vs other organs) Hyp (Hypothalamus vs with full consciousness about the purpose of the Project. The experimental other organs) and Ov (Ovary vs other organs). For each organ DE- design was approved by the Animal Ethic Committee of the University of ncRNAs, RNAcentral identification code (Id), logFC, FDR, Type on ncRNA Milan. Animals were transported, euthanized, sacrificed and organs were and annotation are reported. (XLSX 28 kb) collected according to the European Directive 2010/63/EU on the protection Additional file 4: Distribution of over-expressed ncRNA (DE-ncRNA) of animals used for scientific purposes. (FDR < 0.01 and LogFC> 0) for Pit (Pituitary vs other organs) Hyp (Hypo- thalamus vs other organs) and Ov (Ovary vs other organs). For each Competing interests organ DE-ncRNAs were sorted by categories: long non-coding RNAs The authors declare that they have no competing interests. (lncRNAs), miscellaneous RNA (misc_RNA), precursor_RNA, ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), signal recognition particle RNA SRP_RNA, transfer RNA (tRNA). (DOCX 15 kb) Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Additional file 5: List of organ specific overexpressed miRNA published maps and institutional affiliations. (DOCX 16 kb) Additional file 6: Pathways identified for (experimentally observed) Author details genes targeted by upregulated miRNA expressed in the pituitary, Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle hypothalamus and ovary. (XLSX 148 kb) 2 3 Ricerche, Lodi, Italy. Parco Tecnologico Padano, Lodi, Italy. Dipartimento di Additional file 7: Comparison between A) RNA-Seq and B) Real-time Medicina Veterinaria, Università degli studi di Milano, Milan, Italy. Istituto di PCR data, for 5 miRNAs (miR-141, miR-7-5p, miR-9-5p, miR-124a, miR-10a- Zootecnica, Università Cattolica del Sacro Cuore, Piacenza, Italy. Davies 5p), obtained from each organ: hypothalamus (Hyp), pituitary (Pit) and Research Centre, School of Animal and Veterinary Sciences, University of ovary (Ov) and three replicate (1, 2, 3). (DOCX 70 kb) Adelaide, Roseworthy, Australia. Additional File 8: Novel miRNA mature sequences in fasta format. Fasta Received: 3 July 2017 Accepted: 9 May 2018 headers report the absolute genomic start position of the sequence or the ID of the similar miRNA for novel miRNA detected by similarity to other species (cow, sheep or horse). (TXT 16 kb) References Additional File 9: Novel miRNA precursors sequences in fasta format. 1. Thackray VG, Mellon PL, Coss D. 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Life Sciences; Life Sciences, general; Microarrays; Proteomics; Animal Genetics and Genomics; Microbial Genetics and Genomics; Plant Genetics and Genomics
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Abstract

Background: Molecular regulation of the hypothalamic-pituitary-gonadal (HPG) axis plays an essential role in the fine tuning of seasonal estrus in Capra hircus. Noncoding RNAs (ncRNAs) are emerging as key regulators in sexual development and mammalian reproduction. In order to identify ncRNAs and to assess their expression patterns, along the HPG axis, we sequenced ncRNA libraries from hypothalamus, pituitary and ovary of three goats. Results: Among the medium length noncoding RNAs (mncRNAs) identified, small nucleolar RNAs (snoRNAs) and transfer RNAs (tRNAs) were found to be more abundant in ovary and hypothalamus, respectively. The observed GC content was representative for different classes of ncRNAs, allowing the identification of a tRNA-derived RNA fragments (tRFs) subclass, which had a peak distribution around 32–38% GC content in the hypothalamus. Differences observed among organs confirmed the specificity of microRNA (miRNA) profiles for each organ system. Conclusions: Data on ncRNAs in organs constituting the HPG axis will contribute to understanding their role in the physiological regulation of reproduction in goats. Keywords: miRNA, HPG, Goat, Small-RNA, Reproduction Background Noncoding RNAs (ncRNAs) are involved in a remark- The hypothalamic-pituitary-gonadal (HPG) axis regu- able variety of biological functions. These RNAs are lates reproduction in mammals from fetal development, divided into several families based on their size and through puberty to sexual maturity [1]. The coordin- biogenesis pathways, and act as part of RNA-protein com- ation of peripheral organs with the central nervous plexes in regulating gene expression [4]. Regulatory system ensures that animal physiology is aligned with ncRNAs can be placed in three major classes based on the external environment to optimize reproductive transcript size: small (sncRNAs), medium (mncRNAs) and success [2]. The goat oestrous cycle is accompanied by long noncoding RNAs (lncRNAs) [5]. These different hormonal changes along the HPG axis, that orchestrate ncRNAs are further classified based on sequence or struc- morphological and physiological changes in the ovaries ture conservation, subcellular localization and function, leading to ovulation and preparation of the reproductive association with annotated protein-coding genes and tract for oocyte maturation, sperm transport, fertilization, other DNA elements of known function [6]. Various and embryo implantation [3]. bioinformatic tools are available that use either sequence motifs or structural parameters to detect novel ncRNAs [7]fromsequence data. * Correspondence: stella@ibba.cnr.it Various classes of ncRNAs have roles in promoting Emanuele Capra and Barbara Lazzari contributed equally to this work. the mammalian sexual phenotype [8]. Antisense long Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle non coding RNAs (lncRNAs) may affect the expression Ricerche, Lodi, Italy Parco Tecnologico Padano, Lodi, Italy and function of genes regulating sex determination and Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Capra et al. BMC Genomics (2018) 19:417 Page 2 of 9 gonad development, such as the forkhead box L2 RNA isolation (FOXL2)[9]. The U17 short nucleolar RNA (snoRNA) Total RNA was isolated from each sample using Trizol has been shown to regulate a small non-coding RNA (Invitrogen, Carlsbad, CA) and purified by the NucleoSpin® produced from the Snhg3 introns which influence cellu- miRNA kit (Macherey-Nagel, Germany), following the lar cholesterol trafficking in the ovary, and thus may play protocol recommended by the manufacturer to prepare a role in regulating steroid hormone production and small and large RNA in one fraction (total RNA). RNA postnatal gonadal maturation [10]. The role of miRNAs concentration and quality was determined using an Agilent in modulating the HPG axis is well documented. For 2100 Bioanalyzer (Santa Clara, CA). The isolated RNA was example, in the hypothalamus Lin28/let-7 expression stored at − 80 °C. patterns are associated with the onset of puberty [11]. Lin28a and Lin28b expression decreases during the on- Library preparation and sequencing set of puberty transition and Lin28b expression in the Small noncoding RNA (sncRNA) libraries were gener- ovary may be affected by environmental cues to delay ated using the TruSeq Small RNA Library Preparation puberty and/or follicular development [12]. MiR-361-3p kit according to manufacturer’s instructions (Illumina). is involved in regulating FSH secretion in a pig pituitary The libraries were then pooled (3 goats for 3 organs) cell model [13]. Examination of genome-wide miRNA and purified on a Pippin Prep system (Sage Science, expression in goats suggested that miR-424-5p and miR- MA, USA) to recover two fractions of 125 to 167 nt 29a regulate muscle development [14] and that miRNAs (fraction 1, Illumina adapters included), containing play an important role in endometrial receptivity [15]. It mature miRNAs, and of 168 to 300 nt (fraction 2, was also observed that miRNA differ in their expression Illumina adapters included), containing other ncRNAs, in the three stages of hair follicle cycles in cashmere respectively. The quality and yield after sample prepar- goats [16], as well as in the ovary between pregnant and ation was measured with an Agilent 2200 Tape Station, non-pregnant individuals, with 294 miRNA upregulated High Sensitivity D1000 (Santa Clara, CA). Libraries were and 113 downregulated in pregnant goats [17]. quantified by Real Time PCR with KAPA Library Quan- Although miRNAs and other ncRNAs have shown to tification Kits (Kapa Biosystems, Inc. MA, United play a role in the regulation of tissue development and States). Libraries from fraction 1 and fraction 2 were se- function, little is known about their tissue specification quenced on Miseq desktop sequencer (Illumina) with that to organ functionality along the reproductive axis. 50-base and 150-base single reads, respectively. In the present study, the ncRNA repertoires of hypo- thalamus, pituitary and ovary were investigated in Capra Bioinformatic analysis hircus by use of NGS. All sequences were quality checked with FastQC (http:// www.bioinformatics.babraham.ac.uk/projects/fastqc/) Methods and trimmed with Trimmomatic (minimum sequence Animals and tissue collection quality 30 and minimum sequence length 14) [18]. Three adult female Saanen goats, aged 43.3 ± 3.2 months (mean ± SD) and weighing 55.0 ± 2.3 kg (mean ± SD), ncRNA reared in the same group and on the same farm were ncRNA sequences from fraction 2 from hypothalamus, sacrificed at the end of their productive life. The private pituitary and ovary were collected and separately col- owner agreed to yield them to the present research in- lapsed in silico into three non-redundant datasets with stead of the slaughterhouse with full consciousness the Fastx-Toolkit collapser tool (http://hannonlab.cshl. about the purpose of the Project. edu/fastx_toolkit/). These datasets were compared by The experimental design was approved by the Animal BLASTn to the RNAcentral database sequences [19] and Ethic Committee of the University of Milan. Animals were hits having at least 90% coverage and 90% similarity to transported, anesthetized (Ketamine, 5 mg/kg/IV and the RNAcentral entries were assigned to the RNA class Diazepan 1 mg/kg/IV), sacrificed by receiving a single of the corresponding RNAcentral sequence. Statistical intravenous (IV) bolus injection of a 10 mL solution of analyses were performed with EdgeR [20] and the GLM embutramide, mebezonium iodide and tetracaine hydro- model was applied to identify sequences with differential chloride (Tanax) and organs were collected according to expression among the three datasets. Differential expres- the European Directive 2010/63/EU on the protection of sion analyses across the three organs were run with the animals used for scientific purposes. Samples of the hypo- Bioconductor edgeR package (GLM model, FDR < 0.01 thalamus, pituitary and ovaries were collected from each and LogFC> 1.5). goat. The samples were immediately frozen in liquid ni- GC content of goat ncRNA sequences and entries present trogen and ground to fine powder using mortar and pestle in the small human noncoding RNAs DASHR database and stored at − 80 °C until RNA extraction. (http://lisanwanglab.org/DASHR/smdb.php#tabData)[21], Capra et al. BMC Genomics (2018) 19:417 Page 3 of 9 were calculated with Geecee (http://www.bioinformatics.nl/ Results cgi-bin/emboss/geecee). Libraries preparation NcRNA libraries obtained from hypothalamus, pituitary miRNA data analysis and ovaries have three major size peaks, corresponding miRNA detection and discovery were carried out with to 149, 201 and 268 bp (Fig. 1a). To represent the variety Mirdeep2 on Illumina high quality trimmed sequences of small RNAs in goat, two small RNA fractions from from fraction 1. Capra hircus miRNA sequences available pooled libraries (3 tissues for 3 animals) were selected: i) at MirBase (http://www.mirbase.org/)wereusedtoidentify “fraction 1” containing miRNAs (about 20–30 nt in known miRNA in the trimmed sequences. Known miRNA length) and ii) “fraction 2” containing other ncRNAs from related species (sheep, cow and horse) available at (70–140 nt); Illumina adapters were about 120 bp in size MirBasewerealsousedbyMirdeep2tosupport theidenti- (Fig. 1b). Both fractions were sequenced and analyzed fication of novel goat miRNAs. The Mirdeep2 quantifier separately for their miRNAs and ncRNAs content. Data module was used to quantify expression and retrieve counts are available in the Sequence Reads Archive (SRA), Bio- for the known and novel miRNAs. Differential expression Project accession number, SRP136431. analyses across the three organs were run with the Biocon- ductor edgeR package (FDR < 0.01 and LogFC> 1.5). ncRNA analysis MiRNA cluster analysis was performed with the Genesis Miseq sequencing of fraction 2 (ncRNA 70-140 nt) re- software to identify and visualize patterns within the sulted in 22,309,383 total of raw reads, with an average datasets [22]. MiRNA target prediction was performed by of 2,447,141 reads per sample. FastQC analysis grouped Ingenuity Pathway Analysis (IPA, Ingenuity System, www. trimmed sequences depending on the nucleotide length ingenuity.com). Human homologous miRNAs were ana- and GC content (Additional file 2). In order to classify lyzed with. the sequenced Capra hyrcus ncRNAs according to the microRNA Target filter (IPA) to attribute (experimen- known ncRNA classes available in literature, homology tally observed) target genes. Gene ontology (GO) classifi- searches against the sequence dataset of the RNAcentral cation of miRNA target mRNA was performed according non-coding RNA sequence database [19] were carried to classical GO categories, using the Cytoscape plug-in out (Fig. 2). A high percentage of reads was assigned to ClueGO which integrates GO [23] and enhances small nucleolar RNA (snoRNA), ribosomal RNA (rRNA) biological interpretation of large lists of genes. , transfer RNA (tRNA), lncRNA and signal recognition particle (SRP RNA), while small nuclear (snRNA) and miRNA validationby real time PCR qRT-PCR precursor RNA, ribonuclease P and MRP RNA (Rnase P RNA samples isolated from each organ were retro- RNA and RNAse MRP RNA), miscellaneous RNA (mis- transcribed with miScript II RT Kit following manufac- cRNA), miRNA, antisense RNA (asRNA), guide RNA turer’s instructions (Qiagen, Inc., Valencia, CA USA). (gRNA) and vault RNA (vRNA) were less abundant. The Quantitative Real Time PCR (RT-PCR) was carried out ncRNA size and GC content distributions were consist- on cDNAs with 7900HT Fast Real-Time PCR System ent across all three organs. The majority of the ncRNAs (Applied Biosystems, Carlsbad, California, USA). were in the 60–90 nt size range. There was also a large Reactions were carried out in 10 μl volumes containing number of lnRNAs, snRNAs and Rnase P RNAs reads 1 M of each primer, 2 μl cDNA (see above), and 5 μl2× ranging from 115 to 140 nt long. The distribution of Power SYBR® Green PCR Master Mix (Applied Biosys- gRNAs showed two peaks, corresponding to 60-90 nt tems) according to manufacturer protocols. The primers and 142-151 nt. Different ncRNA classes were distrib- used for chi-miR-141, chi-miR-7, chi-miR-9-5p and chi- uted according to GC content: sequences with low GC miR-10a-5p quantification, were designed using miR- percentage were observed for snoRNA (peak at 32–38%) primer software [24], (Additional file 1). For miR-124a- , snRNA and precursor RNA (peak at 39–42%); a GC 1 quantification, the bta-mir-124a-1 miScript Primer content near 50% was observed for lncRNA (peak at Assay (Qiagen, Inc., Valencia, CA USA) was used. 45–49%) and rRNA, SRP RNA, miscRNA, miRNA, Normalization used the small nucleolar snoRNA as ref- antisense RNA, guide RNA and vault RNA (peak at erence, C/D Box 95 SNORD95 miScript Primer 50–54%); and a high GC content was observed for (Qiagen, Inc., Valencia, CA USA). Negative controls tRNA, RnaseP and Rnase MRP (peak at 60–66%). using water in place of samples were performed along- This highlights that in most cases ncRNA classes are side each reaction. Reactions were run using the cycling characterized by a defined GC content. parameters of 95 °C for 10 min, plus 40 cycles of 95 °C To validate this observation and further explore the for 15 s, and 60 °C for 1 min. Relative expression levels intrinsic properties of ncRNAs, the GC content of en- and significance for each treatment were calculated tries present in the human sncRNAs DASHR database separately using the 2-Ct method [25]. [19], including sequences belonging to different ncRNA Capra et al. BMC Genomics (2018) 19:417 Page 4 of 9 Fig. 1 Small RNA libraries preparation. A Agilent 2100 bioanalyzer profile of a small RNA library obtained from RNA extracted from Hypothalamus, Pituitary and Ovary. B Agilent Tape station profile of a small RNA library fraction obtained by size-selection with pippinprep: miRNA libraries (144 bp), ncRNA libraries (198 and 266 bp). In circle sRNA libraries isolated in fraction 1 and fraction 2: a) 144 bp, b) 198 bp and (c) 266 bp. Illumina adapters were120 bp long classes (rRNA, snoRNA, snRNA, tRNA), was calculated. hypothalamus. MiRNA precursors were under-represented The distribution was similar to that observed for in the pituitary (Fig. 2). Statistical analysis, based on ncRNAs in Capra hircus (Fig. 3). However, ncRNA GC 1549 ncRNAs expressed in three organs, revealed that distribution between goat and human presented some 8, 147 and 94 ncRNAs were differentially expressed be- differences, that could be probably related to the differ- tween pituitary, hypothalamus and ovary (FDR < 0.01) ent dataset used, repository and sequencing data for hu- (Additional file 3). The hypothalamus had a high pro- man and goat respectively. portion of tRNAs, whereas the ovary was enriched for The DASHR database contains the tRNA-derived snoRNAs (Additional file 4). RNA fragments (tRFs), which had a peak at 32–38% GC content. A similar GC content was observed for the miRNA analysis tRNA class in the hypothalamus that was probably asso- Miseq sequencing of fraction 1 (trimmed ncRNA of ciated with the presence of tRFs in this organ (compare about 20-30 nt in length) resulted in 12,592,015 total Fig. 2 and Fig. 3). Expression levels of other ncRNA clas- raw reads, with an average of 1,399,112 reads per sam- ses differed among organs, i.e. miscRNA, snoRNA and ple. The miRNA content in ncRNA libraries was ex- precursor RNA were more abundant in the ovary, plored by bioinformatic analysis of sequenced products lnRNA in the pituitary and tRNA and vRNA in the using the miRDeep2 software. 785 known and putative Capra et al. BMC Genomics (2018) 19:417 Page 5 of 9 Fig. 2 Distribution of different ncRNA classes in function of nucleotide length (nt) and GC content percentage (GC content) for the reads mapped against the RNAcentral database sequence collection. For each category the relative aboundance in each organis was reported (Y axis) miRNAs were identified and quantified in the tested pathway analysis revealed that many genes were involved samples. Among these miRNAs, 402 were already in fibroblast growth factor and epidermal growth factor known in Capra hircus (chi-miRNAs), 222 had hom- response in the pituitary. Pathways related to the ology with known miRNAs from other species and 161 regulation of macromolecule metabolic process, organ were predicted candidate novel miRNAs. After applying development, cellular and developmental processes were a stringent filtering (FDR < 0.01) for each target organ, prevalently targeted by the miRNA found upregulated in 87, 70, and 233 miRNA were identified that were differ- the hypothalamus and ovary (Additional file 6). entially expressed between pituitary, hypothalamus and Differential expression of specific miRNAs in each ovary, respectively. The differential expression was ob- organ was confirmed by qRT-PCR. MiR-141 and miR-7 tained by comparing the expression in one organ versus were highly expressed in the pituitary and miR-9 and the expression in the other two organs (Fig. 4). A list miR-124 highly expressed in the hypothalamus, whereas of organ-specific overexpressed miRNAs is given in miR-10a-5p had the highest level of expression in the Additional file 5. ovary (Additional file 7). Target genes of organ specific upregulated miRNAs found in this study were predicted, and related pathways Discussion identified. 6, 13 and 25 miRNA upregulated in pituitary, Goat mncRNA profiling of three organs according to hypothalamus and ovary targeted 54, 329 and 970 ex- GC content, showed five major peaks (GC content of perimental observed mRNA respectively. The canonical peaks: 32–38%, 39–42%, 45–49%, 50–54%, and 60–66%), Capra et al. BMC Genomics (2018) 19:417 Page 6 of 9 Fig. 3 a total GC content distribution and (b) defined range GC content distribution, calculated with the EMBOSS geecee software on ncRNA entries from the small human noncoding RNAs DASHR database and on ncRNAs (experimentally observed data) for all three organs hypothalamus (Hyp), pituitary (Pit) and ovary (Ov) together in Capra hircus. rRNA, snoRNA, snRNA, tRNA were present in both DASHR database and experimental dataset. tRF-RNA class was present only in the DASHR database. On the Y axis percentage of the relative aboundance of each category of ncRNAs was re- ported. The X axis reports the percentage of GC content for each group leading us to postulate that different mncRNAs have splicing through pre-mRNA secondary structures [32]. specific GC contents. Evaluation of distribution of GC GC content has also been found to influence the content for different classes of small human noncoding function of sncRNA. Short interfering RNA (siRNA) RNAs available in the DASHR database supports our re- GC-content correlates with RNA interference (RNAi) sults. Although GC content was one of the most useful efficiency [33]. GC-content of synonymous codons in features for separating ncRNAs from other genomic ele- coding sequences is proven to have an impact on amino ments [26, 27], we describe a deviation from random acid usage [34]. GC content for each mncRNA class. The relative abundance of the different mncRNA classes The CG content is an important feature that affects was similar in all three caprine organs: snoRNA, rRNA function and stability of RNA: CG rich mRNA is more and tRNA were the most represented, in agreement with efficiently translated, affecting protein products levels quantitative data on the expression landscape of small [28]. GC composition also influences the degradation human noncoding RNA from other tissues available in the rate of mRNAs [29] and lncRNAs [30] and affects DASHR database. stability of RNA secondary structure [31]. It has been In the current version of the database, the distribution suggested that GC content around splice sites affects of the various ncRNAs classes is different for different Capra et al. BMC Genomics (2018) 19:417 Page 7 of 9 Val Gly type of tRNA and tRNA derived fragments were observed to be specifically produced in a controlled fashion in rat brain exposed to ischemia [35]. The level of tRFs was observed to increase when tRF targets decreased with age in rat brain [36]. tRNA-derived small RNAs served as novel signaling molecules in the re- sponse to stress [37, 38]. The high level of tRF found in goat hypothalamus may be important for maintaining a correct epigenetic asset and regulating organ function. SnoRNAs regulate gene expression, playing a central role in ribosome biogenesis. However, many snoRNAs have not been ascribed a function, suggesting that they may have a different cell functionality [39]. Goat hypo- thalamus highly expresses SNORD109A, SNORD114 and SNORD116. Recently, an updated human snoR- NAome based on snoRNAs from RFAM-based predic- tions, generated by the GENCODE consortium, found SNORD116 family and SNORD109 to be specifically overexpressed in neurons [40]. Capra hircus ovary was enriched in many snoRNA. The relative overexpression of SNORD (58, 93, 19, 69, 101, 46, 58, 121A, 58, 19b, 24, 38, 12 and 106) in goat ovary, matched snoRNAs profiling between hypothal- amus and ovary from juvenile female sheep collected in Expression Atlas (http://www.ebi.ac.uk/gxa/home). On the contrary, three of the ovary overexpressed snoRNA in goat SNORD (18, 42 and 25) showed an opposite expression. MiRNAs are regulators of gene expression that exhibit tissue and developmental-specific patterns and contri- bute in maintaining tissue homeostasis [41, 42]. In the present study, several specific miRNAs were predomin- antly expressed in one particular organ. MiR-141, miR200a and miR-7 were expressed in the pituitary gland while miR-124, miR-128 and miR-9 were highly expressed in the hypothalamus. This has also been observed in rodents [41] and in humans [43]. In the present study high levels of expression of miR-10b, miR- 125b, miR-143, miR145, miR199b, miR21 and miR-99a were recorded in the ovary. A recent review identified that these miRNAs were highly expressed in mammalian Fig. 4 Hierarchical clustering obtained from normalized miRNA ovary [44]. count for each replicate (1, 2, 3) in the three organs: hypothalamus (Hyp), pituitary (Pit) and ovary (Ov). A subset of miRNAs showing the highest variance among organs is reported. Red indicates an increase in expression and green a decrease in expression relative to Conclusions the mean expression of 60 miRNAs In summary, this study described the goat (Capra hircus) ncRNA expression profiles in the three organs of the HPG axis. Comparison of these data with similar data tissues. We found that hypothalamus, pituitary and from other species, when it becomes available, will ovary ncRNAs content is specific for each of the goat or- provide insights into the role of different ncRNAs in the gans. The hypothalamus from goat was enriched for reproductive process. Finally, the ncRNA profiling may tRNAs and tRFs whereas ovary had an high level of serve as a reference for further studies investigating the snoRNAs. Intriguingly, goat hypothalamus expressed a peculiarities of goat reproductive physiology, including Gly(GCC) Val(AAC) high level of tRNA and tRNA . The same seasonality in both sexes. Capra et al. BMC Genomics (2018) 19:417 Page 8 of 9 Additional files Authors’ contributions GP, AS, JLW, PAM, BC2, PC conceived and designed the experiments. GP, PC, AS supervised the progress of the project. SF, EC, SC, BC1, AT, gathered Additional file 1: Primer list and sequences used for Real Time samples, prepared hypothalamus and ovary of the goats. BL, EC, AT, SF, PC validation experiment. (XLSX 9 kb) conducted the bioinformatics and statistical analysis. SF, EC conducted the Additional file 2: FastQC analysis result summary for ncRNA sequences experiments. EC, BL, SF, wrote the manuscript. All authors read and approved after trimming process. For each organ an example of the reads the final manuscript. distribution in function of sequence length and per sequence GC content was reported. (DOCX 217 kb) Ethics approval and consent to participate Animals were at the end of their productive career and the private owner Additional file 3: List of differentially expressed ncRNA (DE-ncRNA) agreed to yield them to the present research instead of the slaughterhouse (FDR < 0.01) for Pit (Pituitary vs other organs) Hyp (Hypothalamus vs with full consciousness about the purpose of the Project. The experimental other organs) and Ov (Ovary vs other organs). For each organ DE- design was approved by the Animal Ethic Committee of the University of ncRNAs, RNAcentral identification code (Id), logFC, FDR, Type on ncRNA Milan. Animals were transported, euthanized, sacrificed and organs were and annotation are reported. (XLSX 28 kb) collected according to the European Directive 2010/63/EU on the protection Additional file 4: Distribution of over-expressed ncRNA (DE-ncRNA) of animals used for scientific purposes. (FDR < 0.01 and LogFC> 0) for Pit (Pituitary vs other organs) Hyp (Hypo- thalamus vs other organs) and Ov (Ovary vs other organs). For each Competing interests organ DE-ncRNAs were sorted by categories: long non-coding RNAs The authors declare that they have no competing interests. (lncRNAs), miscellaneous RNA (misc_RNA), precursor_RNA, ribosomal RNA (rRNA), small nucleolar RNA (snoRNA), signal recognition particle RNA SRP_RNA, transfer RNA (tRNA). (DOCX 15 kb) Publisher’sNote Springer Nature remains neutral with regard to jurisdictional claims in Additional file 5: List of organ specific overexpressed miRNA published maps and institutional affiliations. (DOCX 16 kb) Additional file 6: Pathways identified for (experimentally observed) Author details genes targeted by upregulated miRNA expressed in the pituitary, Istituto di Biologia e Biotecnologia Agraria, Consiglio Nazionale delle hypothalamus and ovary. (XLSX 148 kb) 2 3 Ricerche, Lodi, Italy. Parco Tecnologico Padano, Lodi, Italy. Dipartimento di Additional file 7: Comparison between A) RNA-Seq and B) Real-time Medicina Veterinaria, Università degli studi di Milano, Milan, Italy. Istituto di PCR data, for 5 miRNAs (miR-141, miR-7-5p, miR-9-5p, miR-124a, miR-10a- Zootecnica, Università Cattolica del Sacro Cuore, Piacenza, Italy. Davies 5p), obtained from each organ: hypothalamus (Hyp), pituitary (Pit) and Research Centre, School of Animal and Veterinary Sciences, University of ovary (Ov) and three replicate (1, 2, 3). (DOCX 70 kb) Adelaide, Roseworthy, Australia. Additional File 8: Novel miRNA mature sequences in fasta format. Fasta Received: 3 July 2017 Accepted: 9 May 2018 headers report the absolute genomic start position of the sequence or the ID of the similar miRNA for novel miRNA detected by similarity to other species (cow, sheep or horse). (TXT 16 kb) References Additional File 9: Novel miRNA precursors sequences in fasta format. 1. Thackray VG, Mellon PL, Coss D. 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BMC GenomicsSpringer Journals

Published: May 30, 2018

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