Mouse let-7 miRNA populations exhibit RNA editing that is constrained in the 5′-seed/ cleavage/anchor regions and stabilize predicted mmu-let-7a:mRNA duplexes

Department of Chemistry, University of Houston, Houston, Texas 77204, USA.
Genome Research (Impact Factor: 14.63). 07/2008; 18(10). DOI: 10.1101/gr.078246.108
Source: PubMed Central


Massively parallel sequencing of millions of <30-nt RNAs expressed in mouse ovary, embryonic pancreas (E14.5), and insulin-secreting beta-cells (βTC-3) reveals that ∼50% of the mature miRNAs representing mostly the mmu-let-7 family display internal insertion/deletions and substitutions when compared to precursor miRNA and the mouse genome reference sequences. Approximately, 12%–20% of species associated with mmu-let-7 populations exhibit sequence discrepancies that are dramatically reduced in nucleotides 3–7 (5′-seed) and 10–15 (cleavage and anchor sites). This observation is inconsistent with sequencing error and leads us to propose that the changes arise predominantly from post-transcriptional RNA-editing activity operating on miRNA:target mRNA complexes. Internal nucleotide modifications are most enriched at the ninth nucleotide position. A common ninth base edit of U-to-G results in a significant increase in stability of down-regulated let-7a targets in inhibin-deficient mice (Inha−/−). An excess of U-insertions (14.8%) over U-deletions (1.5%) and the presence of cleaved intermediates suggest that a mammalian TUTase (terminal uridylyl transferase) mediated dUTP-dependent U-insertion/U-deletion cycle may be a possible mechanism. We speculate that mRNA target site-directed editing of mmu-let-7a duplex-bulges stabilizes “loose” miRNA:mRNA target associations and functions to expand the target repertoire and/or enhance mRNA decay over translational repression. Our results also demonstrate that the systematic study of sequence variation within specific RNA classes in a given cell type from millions of sequences generated by next-generation sequencing (NGS) technologies (“intranomics”) can be used broadly to infer functional constraints on specific parts of completely uncharacterized RNAs.


Available from: Jayantha B Tennakoon, Jul 08, 2014
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    • "Another reported bias was the overabundance of U insertions as compared with U deletions in the case of isomiR sequencing identified in one study (Reid et al., 2008) but not confirmed in another (Cloonan et al., 2011). The latter difference was explained by the different sequencing platforms used; 454 and Illumina sequencing platforms are more susceptible to insertion or deletion artefacts than SOLiD sequencing. "
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    ABSTRACT: Trinucleotide repeat expansion disorders (TREDs) constitute a group of dominantly inherited neurological diseases that are incurable and ultimately fatal. The underlying cause of TREDs is an expansion of trinucleotide repeats that may occur in the coding and non-coding regions of human genes. MicroRNAs (miRNAs) have emerged as potent regulators of gene expression at the posttranscriptional level. They are involved in a variety of physiological and pathological processes in humans, and the alteration of miRNA expression is considered to be a hallmark of many diseases, including TREDs. This review summarizes the current knowledge regarding the involvement of miRNAs in the pathogenesis of TREDs and the experimentally proven associations between specific miRNAs and particular disorders that have been reported to date.
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    • "We included two closely related miRNAs, miR-10a and miR-10b, which share identical seed sites, but differ by a single nucleotide in the center region. We also included four shifted isomiRs, which are miRNAs produced from the same pre-miRNA as the 'canonical' mature miRNA, but differing in 5′ and/or 3′ end cleavage [13,22]. These isomiRs provide an ideal naturally occurring biological control, as the centered sites are shared between a miRNA and its isomiR, whilst the seed sites differ (Figure 1C). "
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    ABSTRACT: MicroRNAs (miRNAs) bind to mRNAs and target them for translational inhibition or transcriptional degradation. It is thought that most miRNA-mRNA interactions involve the seed region at the 5[prime] end of the miRNA. The importance of seed sites is supported by experimental evidence, although there is growing interest in interactions mediated by the central region of the miRNA, termed centered sites. To investigate the prevalence of these interactions, we apply a biotin pull-down method to determine the direct targets of ten human miRNAs, including four isomiRs that share centered sites, but not seeds, with their canonical partner miRNAs. We confirm that miRNAs and their isomiRs can interact with hundreds of mRNAs, and that imperfect centered sites are common mediators of miRNA-mRNA interactions. We experimentally demonstrate that these sites can repress mRNA activity, typically through translational repression, and are enriched in regions of the transcriptome bound by AGO. Finally, we show that the identification of imperfect centered sites is unlikely to be an artefact of our protocol caused by the biotinylation of the miRNA. However, the fact that there was a slight bias against seed sites in our protocol may have inflated the apparent prevalence of centered site-mediated interactions. Our results suggest that centered site-mediated interactions are much more frequent than previously thought. This may explain the evolutionary conservation of the central region of miRNAs, and has significant implications for decoding miRNA-regulated genetic networks, and for predicting the functional effect of variants that do not alter protein sequence.
    Genome biology 03/2014; 15(3):R51. DOI:10.1186/gb-2014-15-3-r51 · 10.81 Impact Factor
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    • "In the sexually mature chicken ovary library, gga-miR-10a and gga-miR-21 were the two most frequently sequenced miRNAs, and the let-7 miRNA family was another abundant cluster with let-7a being the most abundantly expressed miRNA. The let-7 miRNA family was also expressed abundantly in ovary and oocyte of bovines [28,29,38,39], as well as in murine ovaries and testis [40]. Furthermore, gga-miR-101, gga-miR-1a, gga-miR-146c, gga-miR-148a, gga-miR-126, gga-miR-26a and gga-miR-30d were abundant in our sequencing libraries, as has been shown in other animal gonads [25,27,28]. "
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