Abstract
Members of the Argonaute (Ago) protein family associate with small RNAs and have important roles in RNA silencing. Here, we analysed Ago1- and Ago2-containing protein complexes in human cells. Separation of Ago-associated messenger ribonucleoproteins (mRNPs) showed that Ago1 and Ago2 reside in three complexes with distinct Dicer and RNA-induced silencing complex activities. A comprehensive proteomic analysis of Ago-containing mRNPs identified a large number of proteins involved in RNA metabolism. By using co-immunoprecipitation experiments followed by RNase treatment, we biochemically mapped interactions within Ago mRNPs. Using reporter assays and knockdown experiments, we showed that the putative RNA-binding protein RBM4 is required for microRNA-guided gene regulation.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Argonaute Proteins
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Carboxypeptidases / metabolism
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Cell Line
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Eukaryotic Initiation Factor-2 / genetics
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Eukaryotic Initiation Factor-2 / metabolism*
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Eukaryotic Initiation Factors / genetics
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Eukaryotic Initiation Factors / metabolism*
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Gene Silencing
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Genes, Reporter
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Humans
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Macromolecular Substances
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MicroRNAs / metabolism
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Proteome / analysis*
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins p21(ras)
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RNA, Messenger / metabolism*
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism
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Ribonuclease III / metabolism
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Ribonucleoproteins / metabolism*
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ras Proteins / genetics
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ras Proteins / metabolism
Substances
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AGO1 protein, human
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AGO2 protein, human
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Argonaute Proteins
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Eukaryotic Initiation Factor-2
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Eukaryotic Initiation Factors
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KRAS protein, human
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Macromolecular Substances
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MicroRNAs
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Proteome
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Proto-Oncogene Proteins
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RBM4 protein, human
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RNA, Messenger
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RNA-Binding Proteins
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Ribonucleoproteins
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messenger ribonucleoprotein
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Ribonuclease III
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Carboxypeptidases
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SCPEP1 protein, human
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Proto-Oncogene Proteins p21(ras)
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ras Proteins