MicroRNAs (miRNAs) are very small (~22 nt long) noncoding RNAs that are generated from hairpin precursor transcripts by Dicer-dependent processing. miRNAs likely inhibit translation of mRNAs via imprecise antisense base-pairing. Small interfering RNAs (siRNAs) are similar in size to miRNAs, but they recognize targets by precise complementarity, and elicit RNA-mediated interference (RNAi). To identify as many of the C. elegans miRNA genes as possible, and other kinds of ~22 nt endogenous RNAs expressed in C. elegans, we employed phylogenetic comparisons of noncoding genomic sequences, and sequencing of size-selected cDNA libraries. Among the new small RNAs detected are 21 distinct miRNA sequences that were not previously described in C. elegans. We estimate the size of the C. elegans miRNA gene family to be somewhat more than 100 distinct gene; about 80% of these are conserved in a C. briggsae, and about 30% have apparent homologs in insects and/or vertebrates. We also identified 33 distinct members of a second class of small RNA, which we call tiny noncoding RNAs (tncRNAs). tncRNAs are similar in length to miRNAs, but unlike miRNAs, they are apparently not processed from short hairpin precursors. However, like miRNAs, tncRNAs are transcribed from noncoding genomic sequence, and in some cases, exhibit developmentally regulated expression. tncRNAs also resemble miRNAs in their lack of precise complementarity to any worm mRNA. Therefore, tncRNAs could act in a fashion exemplified by the canonical miRNAs,
lin-4 and
let-7, and recognize RNA targets through imprecise antisense base-pairing. Finally, we identified a third class of ~22 nt RNAs in C. elegans that are produced directly from protein coding sequences and are predicted to have precise antisense complementarity to messenger RNAs. These apparent endogenous siRNAs represent sequences from more than 500 different protein-coding genes, including prominently transposons, kinases, transcription factors, and F-box proteins. Preliminary analysis of worms deficient in Dicer,
alg-1/alg-2 or rde- genes indicates that the accumulation of miRNAs, tncRNAs and siRNAs depend on distinct pathways. These results suggest that RNA-mediated gene regulation and gene silencing may be broadly deployed in normal worm cells through the action of diverse classes of small RNAs.