Proteomic and functional analysis of Argonaute-containing mRNA-protein complexes in human cells.
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.
Höck
Julia
J
Laboratory of RNA Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Weinmann
Lasse
L
Ender
Christine
C
Rüdel
Sabine
S
Kremmer
Elisabeth
E
Raabe
Monika
M
Urlaub
Henning
H
Meister
Gunter
G
D060565:Argonaute Proteins; D002268:Carboxypeptidases / Q000378:metabolism; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing; D017930:Genes, Reporter; D006801:Humans; D046911:Macromolecular Substances; D035683:MicroRNAs / Q000378:metabolism; D020543:Proteome / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
C115848:AGO1 protein, human; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; C117307:KRAS protein, human; D046911:Macromolecular Substances; D035683:MicroRNAs; D020543:Proteome; D011518:Proto-Oncogene Proteins; C473692:RBM4 protein, human; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D043244:Ribonuclease III; D002268:Carboxypeptidases; C436327:SCPEP1 protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins