MicroRNAs (miRNAs) are ~22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs 1 . In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a ~60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex 2,3 . From that duplex, the mature miRNA is incorporated into the silencing complex. Here we identify an alternative pathway for miRNA biogenesis, in which certain debranched introns mimic the structural features of pre-miRNAs to enter the miRNAprocessing pathway without Drosha-mediated cleavage. We call these pre-miRNAs/introns 'mirtrons', and have identified 14 mirtrons in Drosophila melanogaster and another four in Caenorhabditis elegans (including the reclassification of mir-62). Some of these have been selectively maintained during evolution with patterns of sequence conservation suggesting important regulatory functions in the animal. The abundance of introns comparable in size to pre-miRNAs appears to have created a context favourable for the emergence of mirtrons in flies and nematodes. This suggests that other lineages with many similarly sized introns probably also have mirtrons, and that the mirtron pathway could have provided an early avenue for the emergence of miRNAs before the advent of Drosha.While examining sequencing data of small RNAs from D. melanogaster 4 , we observed clusters of small RNAs originating from the outer edges of an annotated 56-nucleotide (56-nt) intron (Fig. 1a). These sets of reads (each read representing an independently sequenced complementary DNA) had properties similar to those observed previously for miRNA:miRNA* duplexes 5 , in that each set had a more consistent 5′ than 3′ terminus, and the two sets were complementary to each other, with the dominantly abundant species of each set forming 2-nt 3′ overhangs when paired to each other. Moreover, the sequence and predicted secondary structure of the intron were conserved in a pattern resembling that of premiRNAs 6 (Fig. 1b, c). We annotated this locus as mir-1003.Despite these clearly miRNA-like properties, semblance to canonical miRNA primary transcripts (pri-miRNAs) stopped abruptly at the borders of the intron. Pairing at the base of the hairpin did not extend beyond the miRNA:miRNA* duplex-that is, beyond the splice sites. In place of extended pairing, which is needed for pri-miRNA cleavage by Drosha (ref.
We sequenced approximately 400,000 small RNAs from Caenorhabditis elegans. Another 18 microRNA (miRNA) genes were identified, thereby extending to 112 our tally of confidently identified miRNA genes in C. elegans. Also observed were thousands of endogenous siRNAs generated by RNA-directed RNA polymerases acting preferentially on transcripts associated with spermatogenesis and transposons. In addition, a third class of nematode small RNAs, called 21U-RNAs, was discovered. 21U-RNAs are precisely 21 nucleotides long, begin with a uridine 5'-monophosphate but are diverse in their remaining 20 nucleotides, and appear modified at their 3'-terminal ribose. 21U-RNAs originate from more than 5700 genomic loci dispersed in two broad regions of chromosome IV-primarily between protein-coding genes or within their introns. These loci share a large upstream motif that enables accurate prediction of additional 21U-RNAs. The motif is conserved in other nematodes, presumably because of its importance for producing these diverse, autonomously expressed, small RNAs (dasRNAs).
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