Intronic microRNA precursors that bypass Drosha processing

Abstract

MicroRNAs (miRNAs) are approximately 22-nucleotide endogenous RNAs that often repress the expression of complementary messenger RNAs. 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 approximately 60-nucleotide pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. 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.

Figure 1. Introns that form pre-miRNAs

a, D. melanogaster mir-1003 with corresponding reads from high-throughput sequencing. The miRNA (red), miRNA* (blue) and splice sites (green lines) are indicated, with predicted secondary structure shown in bracket notation. b, Conservation of mir-1003 across seven Drosophila species,, coloured as in a, and also indicating consensus splice sites (green) and nucleotides differing from D. melanogaster (grey). c, Predicted secondary structures of representative debranched pre-miR-1003 orthologues, coloured as in b. d, Model for convergence of the canonical and mirtronic miRNA biogenesis pathways (see text). e, MicroRNA regulation of luciferase reporters in S2 cells. Plotted is the ratio of repression for wild-type versus mutated sites, normalized to that with the indicated non-cognate miRNA. Bar colour represents the cotransfected miRNA expression plasmid; coloured lines below indicate the cognate miRNA for the specified reporter. Error bars represent the third largest and smallest values from 12 replicates (four independent experiments, each with three transfections; *P < 0.01, **P < 0.0001, Wilcoxon rank-sum test).

Figure 2. Mirtrons are spliced as introns and diced as pre-miRNAs

a, Schematic of splice-site mutations. b, Base pairing between the indicated U1a and mir-1003 RNAs (left), and RT–PCR and northern-blot analyses of mir-1003 variants from a. The miR-1003 bands in lane 2 were attributed to endogenous miRNA. c, Northern blots analysing let-7 and mir-1003 maturation in cells treated with double-stranded RNAs (dsRNAs) corresponding to indicated genes. Shown are results from one membrane, sequentially stripped and probed for let-7 RNA, pre-miR-1003/lariat (probe 1), pre-miR-1003/miR-1003 (probe 2), and U6. Previously validated dsRNAs were used,, except for lariat debranching enzyme (CG7942, which we name ldbr), for which two unique dsRNAs were used. Knockdowns were confirmed by monitoring mRNA level and protein function (Supplementary Fig. S2). Quantification of band intensities is provided (Supplementary Table S3). *Lariat. d, Analysis of mir-1006 processing, as in c.

Figure 3. Emergence and conservation of mirtrons in species with appropriately sized introns

a, Distributions of intron (orange) and pre-miRNA (green) lengths from the indicated species. Introns and pre-miRNAs were binned by length. b, Intron and associated reads of C. elegans mir-62 (ref. 5), coloured as in Fig. 1a. Reads with untemplated nucleotides added at their 3′ terminus are shown below. c, Distributions of pre-miRNA (green) and mirtron (grey) lengths from D. melanogaster and C. elegans. d, Conservation of all 40–90-nt introns (orange) versus mirtrons (grey) from D. melanogaster (percentage identity shared with D. pseudoobscura) and C. elegans (percentage identity shared with C. briggsae).