Discovery of hundreds of mirtrons in mouse and human small RNA data

Abstract

Atypical miRNA substrates do not fit criteria often used to annotate canonical miRNAs, and can escape the notice of miRNA genefinders. Recent analyses expanded the catalogs of invertebrate splicing-derived miRNAs (''mirtrons''), but only a few tens of mammalian mirtrons have been recognized to date. We performed meta-analysis of 737 mouse and human small RNA data sets comprising 2.83 billion raw reads. Using strict and conservative criteria, we provide confident annotation for 237 mouse and 240 human splicing-derived miRNAs, the vast majority of which are novel genes. These comprise three classes of splicing-derived miRNAs in mammals: conventional mirtrons, 59-tailed mirtrons, and 39-tailed mirtrons. In addition, we segregated several hundred additional human and mouse loci with candidate (and often compelling) evidence. Most of these loci arose relatively recently in their respective lineages. Nevertheless, some members in each of the three mirtron classes are conserved, indicating their incorporation into beneficial regulatory networks. We also provide the first Northern validation for mammalian mirtrons, and demonstrate Dicer-dependent association of mature miRNAs from all three classes of mirtrons with Ago2. The recognition of hundreds of mammalian mirtrons provides a new foundation for understanding the scope and evolutionary dynamics of Dicer substrates in mammals.[Supplemental material is available for this article.]Diverse pathways of conserved post-transcriptional gene regulation are mediated by Argonaute proteins and their guide, short RNAs. Among Argonaute-mediated small RNA pathways, the beststudied are the microRNAs (miRNAs). Generally speaking, miRNAs are ;21 to 24-nucleotide (nt) RNAs whose termini are precisely defined, and derive from precursor transcripts bearing one or more inverted repeats or hairpins (Axtell et al. 2011). The first miRNAs emerged from genetic studies of Caenorhabditis elegans developmental mutants (Lee et al. 1993;Reinhart et al. 2000), and were only recognized as noncoding loci upon their cloning. This set the stage for the directed identification of miRNA genes from cloned short RNAs (Lagos-Quintana et al. 2001;Lau et al. 2001;Lee and Ambros 2001). In animals, most miRNAs are generated by stepwise cleavage of primary miRNA transcripts ). These are processed in the nucleus by the Drosha RNase III enzyme to release an ;50-to 80-nt pre-miRNA hairpin, and again in the cytoplasm by a Dicer-class RNase III enzyme to yield a small RNA duplex. One of the strands is preferentially stably incorporated as a singlestranded RNA in an Argonaute (Ago) complex, and guides it to target transcripts (Czech and Hannon 2010).Although bioinformatic strategies have been used to identify miRNA genes Lim et al. 2003;Huang et al. 2007; van der Burgt et al. 2009), these have mostly been superseded by deep sequencing. This is in large part due to the fact that effective computational methods rely on comparative genomics and are ill-suited to identify species-specific miRNAs with reasonable specificit...

Analysis of 13 cell types reveals evidence for the expression of numerous novel primate- and tissue-specific microRNAs

“…1) (PDF images of the predicted precursor structures can be downloaded by following each of the links contained in Dataset S2 or by visiting directly https://cm.jefferson.edu/novelmirnas-2015/). We further postprocessed the identified mature miRNAs and precursors to remove any predicted mature miRNA that (i) is already represented in miRBase release 20 or in the mirtron catalog (28) and/or (ii) colocalized with snRNAs, tRNAs, and rRNAs even though such loci have been previously linked with miRNA production (40)(41)(42)(43). This filtering left us with a collection of 3,494 miRNA precursors of which 213 give rise to two mature miRNA products, one from each arm of the precursor, for a grand total of 3,707 mature miRNAs that satisfied the FDR ≤ 0.05 constraint (Dataset S2).…”

“…In such instances, the corresponding precursor arm is referred to as the "driver" arm. However, recent findings (28,37) suggest that miRNA products from the passenger arm (traditionally referred to as "miRNA*" or "miRNA-star") may also be functionally relevant, and acting similarly to the products from the driver arm (37,50). This observation has rekindled interest in the possibility that a double-stranded miRNA precursor can give rise to two functional miRNA products, one from each arm.…”

“…Initial attempts to characterize the miRNA repertoire of an organism assumed that miRNAs and their precursors are conserved (24)(25)(26), but, since then, it has become evident that genus-specific miRNAs also exist in the fruit fly (Drosophila melanogaster) (27), the mouse (Mus musculus) (28), and the worm (Caenorhabditis elegans) (29). This observation suggests that relying on conservation may underestimate an organism's repertoire of miRNAs.…”

“…A little more than 1,800 human miRNA precursors are listed in release 20 (June 2013) of miRBase (31,32), each giving rise to one or two mature miRNA products. Recent analyses using next-generation sequencing have resulted in the identification of new human and mouse miRNAs (28,(33)(34)(35)(36)(37) and have suggested the existence of tissue-specific miRNAs (33). We reasoned that many more miRNAs are present and can be identified through the analysis of additional samples representing more diverse tissue types.…”

The use of high-throughput sequencing methods for plant microRNA research

“…In recent years, atypical miRNA species such as mirtrons [76][77][78][79][80][81][82][83][84] and reverse complementary miRNA genes 85 were identified based on computational predictions, sRNA-seq data and further experimental validations. Besides, based on sRNA-seq and degradome-seq data, we previously proposed that in plants, the intronic regions of the pre-mRNAs (precursors of mRNAs) might be targeted by specific miRNAs for cleavages.…”