Small RNA expression and strain specificity in the rat

BMC Genomics RSemsearachllarRticNleA expression and strain specificity in the rat Sam EV Linsen Elzo de Wit Ewart de Bruijn Edwin Cuppen 0 Hubrecht Institute-KNAW & University Medical Center Utrecht, Cancer Genomics Center , Utrecht , The Netherlands Background: Digital gene expression (DGE) profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR) from six different rat tissues (spleen, liver, brain, testis, heart, kidney). We describe the expression patterns of known and novel micro (mi)RNAs and piwi-interacting (pi)RNAs. Results: We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strainspecific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters. Conclusions: Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system. - Background miRNAs are ~22 nt-long, single stranded RNA molecules that mediate post-transcriptional regulation of gene expression by directing the RNA-induced Silencing Complex (RISC) to the 3' untranslated region (UTR) of target mRNAs [1,2]. As a result, translation is inhibited and/or the mRNA degraded [3,4]. The target spectrum of a miRNA is mostly defined by the seed, i.e. the 1st or 2nd 7 nt, which hybridizes to the target mRNA [5,6]. miRNAs can both act as developmental switches [7-9] or subtly tune expression, when tight regulation of a gene is required [10,11]. Thousands of mRNAs are expected to be under regulatory control of miRNAs [12,13] and the presence or absence of a single miRNA has been shown to affect, albeit modestly, the level of thousands of proteins [14,15]. Thus, miRNAs form a complex regulatory network affecting the majority of genes. A second developmentally vital class of small RNAs are the piwi-interacting (pi)RNAs [16,17], which play a role in the formation of the germ line. In mammals, these ~27 nt ssRNAs are expressed in the reproductive organs, mainly the testis [18,19], where two types can be distinguished. The pre-pachytene piRNAs, which are repeatand transposon-derived, likely play a role in guiding DNA methylation to repeats, thereby silencing transposons [20] and preventing genome instability. Conversely, the pachytene piRNAs are mostly derived from a selected set of genomic clusters that show a very strong strand bias. The function of these genomic clusters, however, remains elusive [18-21]. To a certain extent, development and homeostasis of organ systems depend on miRNAs [22,23] and piRNAs [24]. The laboratory rat (Rattus norvegicus) is a model organism in which organ-systems physiology has been studied for decades [25]. Recent advances in techniques to genetically modify the rat [26-30] enables detailed analyses of rat physiology at molecular levels. Furthermore, well-established genetic systems, such as congenic, consomic and recombinant inbred lines are versatile tools for studying the effect of genetic variation on quantitative traits such as blood pressure [31] or gene expression [32] (corresponding to quantitative trait loci (QTLs) and expression (e)QTLs, respectively). Comprehensive small RNA inventories and profiles are instrumental in such genetical genomics and systems biological approaches, as they serve as a resource for annotation of the genome. Small RNAs are important players in many regulatory processes and are thus important for understanding disease etiology. The rat small RNA inventory described here will also be important for understanding human disease, since many rat models were selected to reflect clinical symptoms [33]. Conserved expression specificity of miRNAs has been described for a number of organ systems or cell cultures, based on (deep) sequencing approaches [34-36]. Simultaneously, species-specific miRNAs have been identified in closely related species [34,36-38], indicating that miRNAs are evolutionary dynamic. The availability of comprehensive species-specific miRNA profiles of different tissues and organ systems is an important requirement for elucidating the biological roles that miRNAs fulfill. More exhaustive profiling will likely improve existing profiles and increase insight in the basis of quantitative and qualitative variations in miRNA expression. We therefore performed digital gene expression (DGE) profiling of small RNAs from six tissues, i.e. brain, liver, spleen, heart, testis and kidney of the BN-Lx and SHR rat inbred strains, the founder strains of the BXH/HXB recombinant inbred panel [39]. We identified 588 known miRNAs (54 in antisense orientation) and 101 new rat miRNAs, originating from 276 and 61 precursor (pre) miRNA loci, respectively. Thirty-one of these pre-miRNAs were not previously characterized in rat, but were found to be homologous to mouse or human loci; 30 novel candidate pre-miRNA loci do not have an apparent homologue in these species. By generating DGE profiles for liver from three individuals from each strain, we observed strainspecific differential miRNA expression for 4 miRNAs. Finally, we identified thousands of piRNAs in the testis samples. The dataset described here greatly contributes to our understanding of miRNA divergence, variation and expression and may be a valuable resource in evolutionary analyses as well as in the interpretation of regulatory networks and functional genomics experiments in the rat. Results and discussion miRNA identification We collected the small RNA fraction and prepared small RNA sequencing libraries from 6 different tissues (i.e. whole brain, liver, spleen, heart, testis and kidney) from two rat inbred strains (polydactyly-luxate syndrome brown Norway (BN-Lx) and spontaneous hypertensive rats (SHR), adult males). The libraries were sequenced on the SOLiD platform version 2 (ABI), generating 115 million small RNA sequence reads. Of all raw reads, 41.9 million could be mapped to the rat genome (see Additional file 1, Table S1 for individual libraries). The length distribution of the vast majority of small (...truncated)