MicroRNAs and other tiny endogenous RNAs in C. elegans

would inhibit subsequent evolutionary variation in the miRNA sequence. Nonconserved miRNAs could play more species-specific roles or regulate less well-conSummary strained target sequences. Although more that 200 different miRNAs have been identified in plants and aniBackground: MicroRNAs (miRNAs) are small noncod- mals, we do not yet have a comprehensive picture of ing RNAs that are processed from hairpin precursor the scope and spectrum of miRNAs in particular species, transcripts by Dicer. miRNAs probably inhibit translation which miRNAs are evolutionarily conserved, or what of mRNAs via imprecise antisense base-pairing. Small other classes of 22 nt noncoding RNAs function in interfering RNAs (siRNAs) are similar in size to miRNAs, eukaryotic cells. but they recognize targets by precise complementarity Small interfering RNAs (siRNAs) are similar in length and elicit RNA-mediated interference (RNAi). We em- to miRNAs but generally differ from miRNAs in their ployed cDNA sequencing and comparative genomics to origin [17]. siRNAs are produced by Dicer cleavage of a identify additional C. elegans small RNAs with properties long double-stranded RNA into short duplexes of about similar to miRNAs and siRNAs. 21–25 nt in length. Although siRNAs are initially double Results: We found three broad classes of small RNAs stranded [18], the antisense strand probably associates in C. elegans: (1) 21 new miRNA genes (we estimate that with the mRNA target as part of the RNA induced silencing C. elegans contains approximately 100 distinct miRNA complex (RISC) [19–21]. In contrast to siRNAs, miRNAs are genes, about 30% of which are conserved in verte- produced by the processing of a small (approximately 70 brates; (2), 33 distinct members of a class of tiny noncod- nt) single-stranded hairpin precursor transcript [3–6]. ing RNA (tncRNA) genes with transcripts that are similar Although most animal miRNAs are thought to imprein length to miRNAs (approximately 20–21 nt) and that cisely base-pair with their targets and thereby elicit are in some cases developmentally regulated but are translational repression [6, 22–25], it has been shown apparently not processed from a miRNA-like hairpin pre- that if an animal miRNA encounters a target with comcursor and are not phylogenetically conserved; (3) more plete complementarity, it can enter the RNAi pathway than 700 distinct small antisense RNAs, about 20 nt and trigger target cleavage [26, 27]. This suggests that long, that are precisely complementary to protein coding the chief difference between miRNAs and siRNAs in regions of more than 500 different genes and therefore animals lies in their origins (either from an endogenous seem to be endogenous siRNAs. small hairpin RNA or from a bimolecular duplex, respecConclusions: The presence of diverse endogenous tively) and that the outcome of miRNA or siRNA action siRNAs in normal worms suggests ongoing, genome- on a target RNA depends more specifically on the nature wide gene silencing by RNAi. miRNAs and tncRNAs are of the interaction of the small RNA with the target (imprenot predicted to form complete Watson-Crick hybrids cise versus precise complementarity). with any C. elegans RNA target, and so they are likely RNAi-mediated gene silencing has been implicated to regulate the activity of other genes by non-RNAi in surprisingly diverse phenomena of normal cells. In mechanisms. These results suggest that diverse modes plants, some miRNAs naturally contain complete comof small RNA-mediated gene regulation are deployed in plementarity to messenger RNAs and seem to function normal worms. as endogenous siRNAs by directing cleavage of their mRNA targets [13, 28–30]. In the fission yeast, Sacchro