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      Genome-wide identification of Ago2 binding sites from mouse embryonic stem cells with and without mature microRNAs

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          Abstract

          MicroRNAs (miRNAs) are 19-22nt non-coding RNAs that post-transcriptionally regulate mRNA targets. To identify endogenous miRNA binding sites, we performed photo-crosslinking immunoprecipitation using Ago2 antibodies, followed by deep-sequencing of RNAs (CLIP-seq) in mouse embryonic stem cells (mESCs). We also performed CLIP-seq in Dicer −/− mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA-dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3′ untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ∼68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences crosslinked to Ago2 in the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation.

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          Most cited references 60

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          MicroRNAs: target recognition and regulatory functions.

           David Bartel (2009)
          MicroRNAs (miRNAs) are endogenous approximately 23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.
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            Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.

            We predict regulatory targets of vertebrate microRNAs (miRNAs) by identifying mRNAs with conserved complementarity to the seed (nucleotides 2-7) of the miRNA. An overrepresentation of conserved adenosines flanking the seed complementary sites in mRNAs indicates that primary sequence determinants can supplement base pairing to specify miRNA target recognition. In a four-genome analysis of 3' UTRs, approximately 13,000 regulatory relationships were detected above the estimate of false-positive predictions, thereby implicating as miRNA targets more than 5300 human genes, which represented 30% of our gene set. Targeting was also detected in open reading frames. In sum, well over one third of human genes appear to be conserved miRNA targets.
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              The functions of animal microRNAs.

               Victor Ambros (2004)
              MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.
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                Author and article information

                Journal
                101186374
                31761
                Nat Struct Mol Biol
                Nature structural & molecular biology
                1545-9993
                1545-9985
                14 December 2010
                23 January 2011
                February 2011
                1 August 2011
                : 18
                : 2
                : 237-244
                Affiliations
                [1 ]The Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [2 ]Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                [3 ]Computational and Systems Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA
                Author notes
                Correspondence should be addressed to P.A.S. sharppa@ 123456mit.edu
                [4]

                Present address: Salk Institute for Biological Studies, La Jolla, California, USA (A.G.Y.); Howard Hughes Medical Institute and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA (G.X.Z.); Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada (C.B.N.).

                [5]

                These authors contributed equally to this work.

                Article
                nihpa255186
                10.1038/nsmb.1991
                3078052
                21258322

                Users may view, print, copy, download and text and data- mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: http://www.nature.com/authors/editorial_policies/license.html#terms

                Funding
                Funded by: National Cancer Institute : NCI
                Award ID: R01 CA133404-04 ||CA
                Funded by: National Cancer Institute : NCI
                Award ID: P30 CA014051-40 ||CA
                Funded by: National Cancer Institute : NCI
                Award ID: P01 CA042063-25 ||CA
                Categories
                Article

                Molecular biology

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