Dynamic expression of small non-coding RNAs, including novel microRNAs and piRNAs/21U-RNAs, during Caenorhabditis elegansdevelopment
© Kato et al.; licensee BioMed Central Ltd. 2009
Received: 30 January 2009
Accepted: 21 May 2009
Published: 21 May 2009
Small non-coding RNAs, including microRNAs (miRNAs), serve an important role in controlling gene expression during development and disease. However, little detailed information exists concerning the relative expression patterns of small RNAs during development of animals such as Caenorhabditis elegans.
We performed a deep analysis of small RNA expression in C. elegans using recent advances in sequencing technology, and found that a significant number of known miRNAs showed major changes in expression during development and between males and hermaphrodites. Additionally, we identified 66 novel miRNA candidates, about 35% of which showed transcripts from their 'star sequence', suggesting that they are bona fide miRNAs. Also, hundreds of novel Piwi-interacting RNAs (piRNAs)/21U-RNAs with dynamic expression during development, together with many longer transcripts encompassing 21U-RNA sequences, were detected in our libraries.
Our analysis reveals extensive regulation of non-coding small RNAs during development of hermaphrodites and between different genders of C. elegans, and suggests that these RNAs, including novel miRNA candidates, are involved in developmental processes. These findings should lead to a better understanding of the biological roles of small RNAs in C. elegans development.
Proper control of gene expression is required for normal development, health maintenance, and successful reproduction. Until recently it had been believed that gene regulatory networks consisted solely of protein-coding genes, and, in particular, those encoding transcription factors. However, the complete sequencing of many organisms has revealed that only a small fraction of most genomes encodes proteins (reviewed in [1, 2]). On the other hand, recent in-depth genome-wide efforts, including full-length cDNA cloning and tiling microarray analysis, have shown that a large fraction of the remaining non-coding regions are much more extensively transcribed into stable RNAs than previously appreciated (reviewed in [1–3]). Notably, significant portions of these transcripts are small, non-coding RNAs, including microRNAs (miRNAs) and Piwi-interacting RNAs (piRNAs).
miRNAs, first discovered in C. elegans [4–6], negatively regulate gene expression by binding to complementary sequences in the 3' untranslated region of their target mRNAs in an Argonaute-protein-dependent manner (reviewed in ). Mature miRNA products, approximately 22 nucleotides in length, are processed from hairpin-loops of larger primary transcripts. The importance of these RNAs is evidenced by their evolutionary conservation across species and by the many biological events in which they are involved, including cell proliferation, apoptosis and metabolism (reviewed in [8, 9]).
piRNAs, another recently discovered class of small non-coding RNAs that are 24 to 30 nucleotides in length, were found in Drosophila, zebrafish and mammals and so named because they interact with Piwi proteins [10–16]. These proteins, in the Argonaute family, are required for germline development [17, 18] and are important for transposon silencing in the germline of several different organisms [11, 14, 19–21]; this suggests that at least one role of piRNAs is to protect the germline genome against transposons. Indeed, many piRNA sequences map to transposon-like repetitive sequences . Recently, a related class of 21-nucleotide RNAs starting with a uracil (21U-RNA) was identified in C. elegans ; these RNAs were subsequently confirmed to be piRNAs [24–26]. Specifically, C. elegans piwi-related gene (prg) mutants display a dramatic reduction of 21U-RNA expression and a significant up-regulation of the mRNA of Tc3 family transposons with concomitant transposition [24–26].
Previous work has demonstrated that expression of some of these small RNA genes is tightly regulated during development. For example, the expression in C. elegans of the two founding miRNAs, lin-4 and let-7, are specifically up-regulated at the second larval (L2) and the fourth larval (L4) stages, respectively, and are necessary for the normal transition from the first to the second larval stage and from the fourth larval stage to the adult, respectively. Additionally, a Piwi-related protein and numerous piRNAs/21U-RNAs were shown to be most abundant in the young adult stage [24–26]. This implies that Piwi protein and piRNAs/21U-RNAs function in the control of gene expression, in addition to suppressing transposon activity, in germline development. These observations suggest that expression of other miRNAs and piRNAs/21U-RNAs is temporally regulated during development. However, few studies have measured temporal patterns in expression of all these small RNAs in parallel.
Here we use recent advances in high-throughput sequencing technology to quantify the expression of non-coding small RNAs, including miRNAs and piRNAs/21U-RNAs, and demonstrate dynamic and sex-specific expression pattern changes during development of C. elegans. Additionally, we identify many novel miRNA candidates and hundreds of novel piRNAs/21U-RNAs, as well as longer 21U-RNA transcripts encompassing mature 21U-RNAs. These results should lead to a better understanding of the expression and function of small RNAs in C. elegans development.
Results and discussion
Deep sequencing detects the majority of known miRNAs
From our libraries, we detected the expression of 133 of the 154 previously annotated C. elegans miRNAs (miRbase release 11.0; Additional data file 2). While we did not detect 21 of the previously reported miRNAs (we suspect that most of these undetected miRNAs may not actually encode miRNAs at all [23, 29] or may be annotated incorrectly; detailed results are shown in Additional data file 3), we did obtain 125 clones of a very rare miRNA, lsy-6, expressed in only one pair of neurons in the C. elegans head . These findings demonstrate the significant sequencing depth of our survey. Conversely, the maximum number of clones we obtained for a single miRNA was 12,295,951 (miR-58; Additional data file 2), which highlights the high dynamic range of miRNA expression that can be surveyed using deep-sequencing technology such as that from Solexa.
Temporal regulation of miRNA expression during development
About 16% of known miRNAs showed major changes in expression at some point during development (for example, between embryo and the mid-L1 stage; Figure 3a, b). We define here 'a major change' as more than a tenfold difference in the number of reads. For example, the let-7 miRNA exhibited a major increase in expression around the mid-L4 stage, as did one of the let-7 family members, miR-48, from the mid-L3 stage (Figures 2 and 3a). Additionally, another well-characterized miRNA, lin-4, showed a large increase in expression from the mid-L2 stage (Figures 2 and 3a). These observations correspond to previously published results [34, 35] and support the validity and reliability for our small RNA libraries and our analysis.
It is interesting that we were able to clone multiple members of the let-7 and lin-4 families from stages where they were not previously known to be expressed (Additional data file 4). For example, we detected small numbers of clones to both let-7 and lin-4 in embryonic stages, many hours earlier than they had been observed previously. It is unclear if these miRNAs function during these earlier stages, since no embryonic phenotypes are known for let-7 or lin-4 null mutants [6, 36]. Conceivably, this could also represent maternal inheritance or a small bleed-through from the adults to the embryos during preparation.
Of the 24 miRNAs with major changes in expression, some had particularly dynamic expression patterns. For example, miR-71 is dramatically up-regulated from the embryo to the mid-L1 stage and then quickly down-regulated at the mid-L2 stage, and again gradually but significantly up-regulated after the mid-L4 stage (Figure 3a; Additional data file 5). Given its temporal regulation, this miRNA might be involved in control of developmental timing, like lin-4 and let-7. Another interesting case is the expression of miR-77, miR-85, miR-240 and miR-246, which is very low or completely absent in earlier developmental stages but increases after the mid-L4 and young adult stages (Figure 3b; Additional data files 4 and 5), implying a potential role in adult functions like reproduction, metabolism or aging. A recent report by Martinez et al.  also mentioned that some of these miRNAs, including miR-85 and miR-240, are temporally regulated during development, mirroring our results. We highlight additional developmentally regulated miRNAs in Additional data file 4.
Male-specific miRNA expression
Identification and characterization of novel miRNA candidates
miRNA expression cluster analysis
To visualize broad trends in the temporal expression of both previously identified and our newly identified miRNAs, we performed a simple hierarchical clustering. (Figure 7). We found that the 199 miRNAs detectable in our analysis assort into roughly five groups: those expressed primarily at the embryonic stage, those enriched in males, and those primarily expressed in early, middle, and late larval development.
Interestingly, we found that genomically clustered miRNAs are not necessarily co-expressed at the same levels. Some sets of miRNA map to specific chromosomal clusters, as in the case of miR-35 to miR-41, which have redundant functions in embryonic development  and are abundantly expressed in the embryonic stage (Figures 3b and 7). Genomically clustered miRNAs are thought to be transcribed as a single transcript and then individual pre-miRNA are subsequently processed out. We found that although these miRNAs have generally similar expression patterns during development (Figure 7), the absolute expression levels are strikingly different (Additional data file 4). Perhaps, then, clustered miRNAs may be differentially controlled at the transcriptional level and/or during subsequent processing.
Our analysis of the changes of miRNA expression during development may provide helpful information in identifying the target genes for these miRNAs. Coupling this data set with several of the studies describing mRNA expression profiles during development and aging of C. elegans [43, 44] could provide correlations pointing to potential miRNA-target pairs, since changes in expression of miRNAs may cause reciprocal expression patterns of their target genes during development of C. elegans. (Although miRNAs that form imperfect duplexes with their targets inhibit protein production in animals, miRNA binding can also result in degradation of the target mRNA in C. elegans ; indeed, microarray analysis has proven to be an effective way to find genes modulated by miRNAs .)
Expression of piRNAs/21U-RNAs during development and in the germline
Another class of C. elegans non-coding small RNAs, 21U-RNAs, have important functions in transposon silencing in the germline and maturation of gametes [24–26]. More than 15,000 unique 21U-RNA sequences have been reported in C. elegans, the vast majority of which map to either intergenic or intronic regions on chromosome IV [23, 25]. As expected from their function in germline development, our results confirmed recent studies that show prominent accumulation of 21U-RNAs in the young adult stage (Figure 1; Additional data files 1 and 9) [24–26].
Approximately 44% of known 21U-RNAs on chromosome IV are genomically clustered within 10 bp with other 21U-RNAs (see below), implying that expression of 21U-RNAs in each cluster is controlled in a similar manner, and one would expect that these clustered 21U-RNAs might show similar changes in expression in both male and hermaphrodite germlines compared to 21U-RNAs mapping outside the clusters. Interestingly, though, we did not detect common patterns in expression of 21U-RNAs in the clusters; that is, 21U-RNA abundance was routinely different for 21U-RNAs in the same cluster, although 21U-RNAs in a genomic cluster appears to be transcribed from the same strand (data not shown).
Identification and characterization of additional piRNA/21U-RNA sequences
While we have not shown that these RNAs associate with Piwi proteins like PRG-1, we suspect that these are very likely to be novel piRNAs/21U-RNAs for several reasons: first, these RNAs are abundantly expressed in the L4 and young adult stages (Additional data file 12; consistent with known 21U-RNAs); second, they are transcribed from the same two distinct regions of chromosome IV as known 21U-RNAs (Additional data file 12); third, they contain the core motif associated with bone fide 21U-RNAs; and fourth, most of them partially overlap with known or other novel 21U-RNAs (see below). Also, approximately 8% of these novel 21U-RNAs were detectable in other libraries obtained by 454 sequencing from different biological sources (ADL and FS, unpublished result).
Identification of larger reads corresponding to piRNAs/21U-RNAs
Our analysis reveals extensive regulation of small, non-coding RNAs during development of C. elegans hermaphrodites and in males, and suggests that these RNAs are involved in developmental processes. Our results also illustrate the extreme diversity of miRNA and piRNA expression in C. elegans. In addition, our deep sequencing approach revealed the presence of tens more miRNAs and hundreds more piRNAs than were previously known. Since the information content of the genome is more complex than previously imagined - for example, most of both strands of the genome appear to be transcribed in human , and approximately 80% of transcripts map to unannotated regions  - it seems likely that additional non-coding RNA genes remain to be discovered and characterized in other animals as well. For instance, in our study, numerous sequence variants of miRNAs were found corresponding to their hairpin sequences, which include many 'star sequences' (Additional data file 3). Identification of further transcripts and their biological roles will lead to a better understanding of animal biology and will shed light on control of gene expression during development and disease.
Materials and methods
C. elegansstrains and small RNA purification
Wild-type N2 strains were cultured under standard conditions  at 20°C and used to prepare RNAs from each developmental stage (time after stage L1: mid-L1 (4 h), mid-L2 (14 h), mid-L3 (25 h), mid-L4 (36 h); and young adult (48 h). RNAs enriched for small RNA species (less than 200 nucleotides) were prepared using the mirVana miRNA Isolation kit (Ambion/Applied Biosystems, Austin, TX, USA) with the small RNA enrichment procedure. For library preparation from young adult males, dpy-28 (y1);him-8 (e1489) double mutants cultured at 23°C were used to obtain male populations after backcrossing six times to wild-type N2, and RNAs were purified at 40 h after stage L1. him-8 (e1489) mutants produce XO males and XXX hermaphrodites at 37% and 6% frequency, respectively, in addition to XX hermaphrodites . However, XX and XXX hermaphrodites can not survive at 23°C in the dpy-28 (y1) background , and the resulting surviving population of dpy-28;him-8 double mutants is almost all XO males at this temperature. For validating novel miRNA expression, total RNAs were isolated from N2 wild-type worms, alg-1 (gk214) mutants and N2 wild-type worms on both L4440 (empty vector) and alg-1 RNAi at the young adult stage.
cDNA library preparation and sequencing
cDNA libraries for small RNAs were made from 10 μg of RNA from an enriched small RNA fraction using the DGE-Small RNA Sample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. The same amount of cDNA was sequenced on a Genetic Analyzer from Illumina. The data from the miRNA reads we mentioned above were uploaded to the Genome Expression Omnibus database together with the raw Solexa sequence results [GEO:GSE13339]. The 66 novel miRNA candidates and the 552 unique piRNAs/21U-RNAs have GenBank accession numbers (shown in Additional data files 7 and 11).
The expression of some of the known miRNAs were confirmed by quantitative RT-PCR using a TaqMan Small RNA Assay (Applied Biosystems, Foster City, CA, USA) with the RNAs at concentrations of 0.4 ng/μl (enriched small RNAs) and 2 ng/μl (total RNAs), according to the manufacture's instruction. For validating the expression of novel miRNA candidates, 10 ng/μl of total RNAs was used, and the results were normalized to the expression level of U18. The results were further confirmed using independently prepared RNA samples.
Computational data analysis
The number of sequence reads for miRNAs and 21U-RNAs was assessed from the raw sequence data from Solexa sequencing using perfect sequence matching to known miRNAs (miRBase release 11.0) and 21U-RNAs  (Additional data files 2, 4 and 9). For examining the proportion of each non-coding RNA species, including rRNAs, tRNAs, snRNAs, and snoRNAs, sequence reads that matched to the C. elegans genome (WS190) were extracted by the SOAP program (a maximum of 2 bp mismatches were allowed in the alignment) , and the number of sequence reads perfectly corresponding to each RNA species was determined using BLASTN against a database of non-coding RNAs from WormBase . To compare the differential expression of small RNAs across development, the number of reads in each sample was normalized to the total number of reads that matched to the C. elegans genome in each sample. The Cluster 3.0 program was used to cluster the miRNAs (after normalizing each gene's expression vector to have a 2-norm of 1). The Java TreeView program  was then used to visualize these clusters. The miRDeep program  was used for finding novel miRNA candidates, and the RNA fold program was used for predicting secondary structure of primary miRNA transcripts of novel miRNAs.
Additional data files
The following additional data are available with the online version of this paper: the total number of sequence reads and number of reads of each non-coding RNA species in each sample (Additional data file 1); raw data showing the number of miRNA reads in each developmental stage of hermaphrodites and in young adult males (Additional data file 2); sequence variants expressed from miRNA hairpins (Additional data file 3); normalized data of the number of miRNA reads by the total number of reads that matched to the C. elegans genome (Additional data file 4); confirmation of miRNA expression changes during development of hermaphrodites and in young adult males using quantitative RT-PCR (Additional data file 5); the correlation between miRNA expression levels in males and hermaphrodites (Additional data file 6); a list of novel miRNA candidates (Additional data file 7); the number of reads of novel miRNA candidates in each sample (Additional data file 8); the number of known 21U-RNA reads in each sample (Additional data file 9); sequence of 21nt-U-RNA reads and their chromosomal position (Additional data file 10); sequence of novel 21U-RNAs (Additional data file 11); changes in expression of novel 21U-RNAs during development and their position on chromosome IV (Additional data file 12); a list of all 21U-RNA longer transcripts detected in our library (Additional data file 13).
We thank Ghia Euskirchen for help with Solexa sequencing and Valerie Reinke for critical reading of this manuscript. We also thank the CGC for strains. MK was partially supported by a postdoctoral fellowship from the Uehara Memorial Foundation; FS was supported by grants from the NIH to the modENCODE consortium (RFA-HG-06-006).
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