Chiang, HR, Schoenfeld, LW, Ruby, JG, Auyeung, VC, Spies, N, Baek, D et al.. Mammalian microRNAs: experimental evaluation of novel and previously annotated genes. Genes Dev 24: 992-1009

Whitehead Institute for Biomedical Research, Cambridge, Massachusetts 02142, USA.
Genes & development (Impact Factor: 10.8). 04/2010; 24(10):992-1009. DOI: 10.1101/gad.1884710
Source: PubMed


MicroRNAs (miRNAs) are small regulatory RNAs that derive from distinctive hairpin transcripts. To learn more about the miRNAs of mammals, we sequenced 60 million small RNAs from mouse brain, ovary, testes, embryonic stem cells, three embryonic stages, and whole newborns. Analysis of these sequences confirmed 398 annotated miRNA genes and identified 108 novel miRNA genes. More than 150 previously annotated miRNAs and hundreds of candidates failed to yield sequenced RNAs with miRNA-like features. Ectopically expressing these previously proposed miRNA hairpins also did not yield small RNAs, whereas ectopically expressing the confirmed and newly identified hairpins usually did yield small RNAs with the classical miRNA features, including dependence on the Drosha endonuclease for processing. These experiments, which suggest that previous estimates of conserved mammalian miRNAs were inflated, provide a substantially revised list of confidently identified murine miRNAs from which to infer the general features of mammalian miRNAs. Our analyses also revealed new aspects of miRNA biogenesis and modification, including tissue-specific strand preferences, sequential Dicer cleavage of a metazoan precursor miRNA (pre-miRNA), consequential 5' heterogeneity, newly identified instances of miRNA editing, and evidence for widespread pre-miRNA uridylation reminiscent of miRNA regulation by Lin28.

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    • "High-throughput sequencing data shows that some pre-miRNAs produce a single dominant mature miRNA from one arm, with a small proportion of reads, even no reads, originating from the opposite arm. Recently, studies have found that dominant miRNAs can switch from one of the two pre-miRNA arms to the other in different tissues and at different times of development (Chiang et al., 2010; Grimson et al., 2008; Ro et al., 2007), even in orthologous miRNAs in different species (Griffiths-Jones et al., 2011; Li et al., 2010, 2011; Marco et al., 2010). To explore armswitching miRNAs during the course of hESC differentiation into RPE cells, we first mapped the reads onto the human genome with consideration to the positions of pre-miRNAs and mature miRNAs. "
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    ABSTRACT: Embryonic stem cells (ESCs) and induced pluripotent stem cells can be induced to differentiate into retinal pigment epithelium (RPE). miRNAs have been characterized and found playing important roles in the differentiation process of ESCs, but their length and sequence heterogeneity (isomiRs), and their non-canonical forms of miRNAs are underestimated or ignored. In this report, we found some non-canonical miRNAs (dominant isomiRs) in all differentiation stages, and 27 statistically significant editing sites were identified in 24 different miRNAs. Morever, we found marked major-to-minor arm-switching events in 14 pre-miRNAs during the hESC to RPE cell differentiation phases. Our study for the first time reports exploring the variability of miRNA expression during the differentiation of hESCs into RPE cells and the results show that miRNA variability is a ubiquitous phenomenon in the ESC differentiation. Copyright © 2015. Published by Elsevier B.V.
    Gene 05/2015; 569(2). DOI:10.1016/j.gene.2015.05.060 · 2.14 Impact Factor
    • "Expression of sRNAs from mRNAs did not correlate with mRNA expression (Figure S4J). As reported before (Chiang et al., 2010), the more abundant sRNA preferentially arises from the 5 0 arm of the hairpin. This holds for known miRNAs and becomes more pronounced for the entire set of active sites (Figure 4D). "
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    ABSTRACT: The endoribonuclease Dicer is known for its central role in the biogenesis of eukaryotic small RNAs/microRNAs. Despite its importance, Dicer target transcripts have not been directly mapped. Here, we apply biochemical methods to human cells and C. elegans and identify thousands of Dicer-binding sites. We find known and hundreds of additional miRNAs with high sensitivity and specificity. We also report structural RNAs, promoter RNAs, and mitochondrial transcripts as Dicer targets. Interestingly, most Dicer-binding sites reside on mRNAs/lncRNAs and are not significantly processed into small RNAs. These passive sites typically harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression. We show that passive sites can sequester Dicer and reduce microRNA expression. mRNAs with passive sites were in human and worm significantly associated with processing-body/granule function. Together, we provide the first transcriptome-wide map of Dicer targets and suggest conserved binding modes and functions outside of the miRNA pathway.
    Cell 11/2014; 159(5):1153-67. DOI:10.1016/j.cell.2014.10.040 · 32.24 Impact Factor
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    • "Based on several considerations, we believe that most of the inefficiently processed pri-miRNAs reported in our study resemble bona fide Microprocessor targets that are processed at a very low rate. First, we performed small RNA sequencing (RNA-seq) and restricted our analysis to pri-miRNAs with robust evidence for authentic expressed miRNA hairpins according to criteria in Chiang et al. (2010) as specified in the Results and Experimental Procedures. Second, our motif analysis revealed a distinct set of sequence features that are shared by efficiently and inefficiently processed miRNAs but are not found in pseudohairpins, like the enrichment of GC dinucleotides in the 5 0 stem of pre-miRNA (Figures 6 and S7). "
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    ABSTRACT: In miRNA biogenesis, pri-miRNA transcripts are converted into pre-miRNA hairpins. The in vivo properties of this process remain enigmatic. Here, we determine in vivo transcriptome-wide pri-miRNA processing using next-generation sequencing of chromatin-associated pri-miRNAs. We identify a distinctive Microprocessor signature in the transcriptome profile from which efficiency of the endogenous processing event can be accurately quantified. This analysis reveals differential susceptibility to Microprocessor cleavage as a key regulatory step in miRNA biogenesis. Processing is highly variable among pri-miRNAs and a better predictor of miRNA abundance than primary transcription itself. Processing is also largely stable across three cell lines, suggesting a major contribution of sequence determinants. On the basis of differential processing efficiencies, we define functionality for short sequence features adjacent to the pre-miRNA hairpin. In conclusion, we identify Microprocessor as the main hub for diversified miRNA output and suggest a role for uncoupling miRNA biogenesis from host gene expression.
    Cell Reports 10/2014; 9(2). DOI:10.1016/j.celrep.2014.09.007 · 8.36 Impact Factor
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