Ameres, S. L. et al. Target RNA-directed trimming and tailing of small silencing RNAs. Science 328, 1534-1539

Howard Hughes Medical Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Science (Impact Factor: 33.61). 06/2010; 328(5985):1534-9. DOI: 10.1126/science.1187058
Source: PubMed


In Drosophila, microRNAs (miRNAs) typically guide Argonaute1 to repress messenger RNA (mRNA), whereas small interfering RNAs (siRNAs) guide Argonaute2 to destroy viral and transposon RNA. Unlike siRNAs, miRNAs rarely form extensive numbers of base pairs to the mRNAs they regulate. We find that extensive complementarity between a target RNA and an Argonaute1-bound miRNA triggers miRNA tailing and 3'-to-5' trimming. In flies, Argonaute2-bound small RNAs--but not those bound to Argonaute1--bear a 2'-O-methyl group at their 3' ends. This modification blocks target-directed small RNA remodeling: In flies lacking Hen1, the enzyme that adds the 2'-O-methyl group, Argonaute2-associated siRNAs are tailed and trimmed. Target complementarity also affects small RNA stability in human cells. These results provide an explanation for the partial complementarity between animal miRNAs and their targets.

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Available from: Stefan Ameres, Oct 10, 2015
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    • "Uridylation has been coupled to target-dependent de-stabilization of small RNAs (Ameres et al., 2010), whereas adenylation has recently been shown to be involved in the clearance of maternal miRNAs (Lee et al., 2014). We therefore checked non-templated nucleotide addition in all bovine samples. "
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    ABSTRACT: Germ cells of most animals critically depend on piRNAs and Piwi proteins. Surprisingly, piRNAs in mouse oocytes are relatively rare and dispensable. We present compelling evidence for strong Piwi and piRNA expression in oocytes of other mammals. Human fetal oocytes express PIWIL2 and transposon-enriched piRNAs. Oocytes in adult human ovary express PIWIL1 and PIWIL2, whereas those in bovine ovary only express PIWIL1. In human, macaque, and bovine ovaries, we find piRNAs that resemble testis-borne pachytene piRNAs. Isolated bovine follicular oocytes were shown to contain abundant, relatively short piRNAs that preferentially target transposable elements. Using label-free quantitative proteome analysis, we show that these maturing oocytes strongly and specifically express the PIWIL3 protein, alongside other, known piRNA-pathway components. A piRNA pool is still present in early bovine embryos, revealing a potential impact of piRNAs on mammalian embryogenesis. Our results reveal that there are highly dynamic piRNA pathways in mammalian oocytes and early embryos.
    Cell Reports 03/2015; 10(12):2069–2082. DOI:10.1016/j.celrep.2015.02.062 · 8.36 Impact Factor
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    • "Future studies should answer whether nonrandom miRNA release has a biological impact on gene regulation, which likely depends on multiple additional factors. Indeed, the level and function of mature miR- NAs are the result of different rates of both miRNA and target transcription, processing location, and turnover (Ameres et al., 2010; Baccarini et al., 2011). "
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    ABSTRACT: Functional biomolecules, including small noncoding RNAs (ncRNAs), are released and transmitted between mammalian cells via extracellular vesicles (EVs), including endosome-derived exosomes. The small RNA composition in cells differs from exosomes, but underlying mechanisms have not been established. We generated small RNA profiles by RNA sequencing (RNA-seq) from a panel of human B cells and their secreted exosomes. A comprehensive bioinformatics and statistical analysis revealed nonrandomly distributed subsets of microRNA (miRNA) species between B cells and exosomes. Unexpectedly, 3' end adenylated miRNAs are relatively enriched in cells, whereas 3' end uridylated isoforms appear overrepresented in exosomes, as validated in naturally occurring EVs isolated from human urine samples. Collectively, our findings suggest that posttranscriptional modifications, notably 3' end adenylation and uridylation, exert opposing effects that may contribute, at least in part, to direct ncRNA sorting into EVs.
    Cell Reports 09/2014; 8(6). DOI:10.1016/j.celrep.2014.08.027 · 8.36 Impact Factor
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    • "Yet, it is largely unknown, particularly in mammals, how cells control the quality of this elaborate and thus error-prone process . One quality control pathway for miRNAs and siRNAs in plants or flies is via decay of small RNAs that lack 2 0 -O-methylation in their 3 0 ends from Agos by noncanonical TUTs, such as HESO1 or MUT68 (Ameres et al., 2010; Ibrahim et al., 2010; Ren et al., 2012; Zhao et al., 2012). However, mammalian miRNAs are not methylated, and the relevance of this surveillance pathway for mammals is not apparent. "
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    ABSTRACT: MicroRNAs (miRNAs) are essential for regulation of gene expression. Though numerous miRNAs have been identified by high-throughput sequencing, few precursor miRNAs (pre-miRNAs) are experimentally validated. Here we report a strategy for constructing high-throughput sequencing libraries enriched for full-length pre-miRNAs. We find widespread and extensive uridylation of Argonaute (Ago)-bound pre-miRNAs, which is primarily catalyzed by two terminal uridylyltransferases: TUT7 and TUT4. Uridylation by TUT7/4 not only polishes pre-miRNA 3' ends, but also facilitates their degradation by the exosome, preventing clogging of Ago with defective species. We show that the exosome exploits distinct substrate preferences of DIS3 and RRP6, its two catalytic subunits, to distinguish productive from defective pre-miRNAs. Furthermore, we identify a positive feedback loop formed by the exosome and TUT7/4 in triggering uridylation and degradation of Ago-bound pre-miRNAs. Our study reveals a pre-miRNA surveillance system that comprises TUT7, TUT4, and the exosome in quality control of miRNA synthesis.
    Molecular Cell 08/2014; 55(6). DOI:10.1016/j.molcel.2014.07.017 · 14.02 Impact Factor
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