Computational and experimental identification of mirtrons in Drosophila melanogaster and Caenorhabditis elegans

Abstract

Mirtrons are intronic hairpin substrates of the dicing machinery that generate functional microRNAs. In this study, we describe experimental assays that defined the essential requirements for entry of introns into the mirtron pathway. These data informed a bioinformatic screen that effectively identified functional mirtrons from the Drosophila melanogaster transcriptome. These included 17 known and six confident novel mirtrons among the top 51 candidates, and additional candidates had limited read evidence in available small RNA data. Our computational model also proved effective on Caenorhabditis elegans, for which the identification of 14 cloned mirtrons among the top 22 candidates more than tripled the number of validated mirtrons in this species. A few low-scoring introns generated mirtron-like read patterns from atypical RNA structures, but their paucity suggests that relatively few such loci were not captured by our model. Unexpectedly, we uncovered examples of clustered mirtrons in both fly and worm genomes, including a <8-kb region in C. elegans harboring eight distinct mirtrons. Altogether, we demonstrate that discovery of functional mirtrons, unlike canonical miRNAs, is amenable to computational methods independent of evolutionary constraint.[Supplemental material is available for this article. Small RNA data have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/). A full list of accession numbers can be found in Supplemental Table S1.]Canonical microRNAs (miRNAs) are ;22-nucleotide (nt) regulatory RNAs derived from inverted repeat transcripts whose biogenesis involves a defined series of processing events (Kim et al. 2009). Primary-miRNA (pri-miRNA) transcripts are first cleaved by the nuclear RNase III enzyme Drosha (also known as RNASEN) to yield pre-miRNA hairpins. Following their cytoplasmic export via exportin 5, pre-miRNAs are cleaved on their terminal loop side by a Dicer-class RNase III enzyme to release miRNA/miRNA* duplexes. One side of the duplex, designated the mature miRNA, is preferentially transferred into an Argonaute protein and guides it to regulate target transcripts. Its partner miRNA* strand is inferred to be preferentially degraded on account of its lower steady-state accumulation, although miRNA* species may still be transferred into Argonaute proteins and have regulatory activities. Since RNase III enzymes typically cleave substrates leaving signature 2-nt 39 overhangs, an appropriate geometry of cloned small RNA duplex ends provides evidence for their transit via a Drosha-Dicer pathway (Ambros et al. 2003;Friedlander et al. 2008;Berezikov et al. 2010;Chiang et al. 2010).Since thousands of miRNAs are now known (Griffiths-Jones et al. 2008), one might presume that sufficient information exists to segregate bona fide miRNA genes from bulk genomic hairpins. Although bioinformatic strategies can enrich for genuine miRNA genes, the number of plausible pri-miRNA hairpins in a typical animal genome exceeds the number of confirmed miRNA hairpins by se...

High-throughput sequencing reveals extraordinary fluidity of miRNA, piRNA, and siRNA pathways in nematodes

“…However, some miRNAs are, instead, derived from short intronic hairpins called mirtrons during splicing, thereby bypassing Drosha cleavage (Okamura et al 2007;Ruby et al 2007). We found that out of the 15 C. elegans annotated mirtrons (Chung et al 2011), only miR-62, embedded in the third intron of ugt-50, is conserved in the other three nematodes (Fig. 2E).…”

Adaptive evolution of testis-specific, recently evolved, clustered miRNAs inDrosophila

“…We expanded our recently published collection of conditional UAS-DsRedmiRNA expression transgenes (Bejarano et al 2012) by generating 28 additional transgenic lines (Supplemental Tables 4, 5), mostly composed of newly evolved miRNAs that we had annotated more recently (Berezikov et al 2011;Chung et al 2011). We then selected all the transgenes for newly evolved miRNAs and performed a systematic screen of all the independent insertions for each construct against nine Gal4 drivers, including ubiquitous (da-Gal4), eye specific (GMR-Gal4, ey-Gal4), wing specific (Sd-Gal4), wing and notum (1096-Gal4), anterior posterior compartment boundary (dpp-Gal4 and ptc-Gal4), notum specific (Eq-Gal4), and neuron specific (elav-Gal4).…”

Common and distinct patterns of terminal modifications to mirtrons and canonical microRNAs

“…However, the depth of available data permitted meaningful conclusions to be drawn (Berezikov et al 2011), since we recovered hundreds to thousands of 5p reads for most of the 31 annotated conventional mirtrons (Chung et al 2011) in an aggregate set of 230 Drosophila small RNA libraries (Data set 1). The 59 ends of mirtron-5p reads exhibited a very high rate of fidelity, since 28 of these exhibited >90% precision of the initiating nucleotide, and 23 of those were defined with >95% precision (Fig.…”