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.

SUMMARY Thousands of long intervening non-coding RNAs (lincRNAs) have been identified in mammals. To better understand the evolution and functions of these enigmatic RNAs, we used chromatin marks, poly(A)-site mapping and RNA-Seq data, to identify more than 550 distinct lincRNAs in zebrafish. Although these shared many characteristics with mammalian lincRNAs, only 29 had detectable sequence similarity with putative mammalian orthologs, typically restricted to a single short region of high conservation. Other lincRNAs had conserved genomic locations without detectable sequence conservation. Antisense reagents targeting conserved regions of two zebrafish lincRNAs caused developmental defects. Reagents targeting splice sites caused the same defects and were rescued by adding either the mature lincRNA or its human or mouse ortholog. Our study provides a roadmap for identification and analysis of lincRNAs in model organisms and shows that lincRNAs play crucial biological roles during embryonic development with functionality conserved despite limited sequence conservation.