MicroRNAs (miRNAs) are ∼22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs1. 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* duplex2,3. 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 miRNA-processing 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.
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We are grateful to P. Sharp, T. Baker and members of the Bartel laboratory for discussions. We thank W. Johnston for assistance with molecular cloning, E. Lai for contributing small-RNA-derived cDNAs for sequencing, the Drosophila genome sequencing community and the UCSC genome browser staff for unpublished alignments, P. Zamore and R. Green for dsRNA plasmids, S. Cohen for GFP and firefly luciferase Drosophila expression plasmids, and D. Sabitini for pMT-puro. This work was supported by the NIH. C.H.J. is a NSF graduate research fellow. D.P.B. is an investigator of the Howard Hughes Medical Institute. Small RNA sequences were deposited in the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/), accessions GPL5061 and GSE7448.
Author Contributions J.G.R. performed the computational analysis. C.H.J. performed the experimental analysis. All authors contributed to the design of the study and preparation of the manuscript.
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Ruby, J., Jan, C. & Bartel, D. Intronic microRNA precursors that bypass Drosha processing. Nature 448, 83–86 (2007). https://doi.org/10.1038/nature05983
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