TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing.
MicroRNAs (miRNAs) are generated by a two-step processing pathway to yield RNA molecules of approximately 22 nucleotides that negatively regulate target gene expression at the post-transcriptional level. Primary miRNAs are processed to precursor miRNAs (pre-miRNAs) by the Microprocessor complex. These pre-miRNAs are cleaved by the RNase III Dicer to generate mature miRNAs that direct the RNA-induced silencing complex (RISC) to messenger RNAs with complementary sequence. Here we show that TRBP (the human immunodeficiency virus transactivating response RNA-binding protein), which contains three double-stranded, RNA-binding domains, is an integral component of a Dicer-containing complex. Biochemical analysis of TRBP-containing complexes revealed the association of Dicer-TRBP with Argonaute 2 (Ago2), the catalytic engine of RISC. The physical association of Dicer-TRBP and Ago2 was confirmed after the isolation of the ternary complex using Flag-tagged Ago2 cell lines. In vitro reconstitution assays demonstrated that TRBP is required for the recruitment of Ago2 to the small interfering RNA (siRNA) bound by Dicer. Knockdown of TRBP results in destabilization of Dicer and a consequent loss of miRNA biogenesis. Finally, depletion of the Dicer-TRBP complex via exogenously introduced siRNAs diminished RISC-mediated reporter gene silencing. These results support a role of the Dicer-TRBP complex not only in miRNA processing but also as a platform for RISC assembly.
Chendrimada
Thimmaiah P
TP
The Wistar Institute, 3601 Spruce Street, Philadelphia, Pennsylvania 19104, USA.
Gregory
Richard I
RI
Kumaraswamy
Easwari
E
Norman
Jessica
J
Cooch
Neil
N
Nishikura
Kazuko
K
Shiekhattar
Ramin
R
D060565:Argonaute Proteins; D002460:Cell Line; D015852:Eukaryotic Initiation Factor-2; D020868:Gene Silencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D035683:MicroRNAs / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D056918:Nuclear Receptor Coactivators; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D047908:Intracellular Signaling Peptides and Proteins; D035683:MicroRNAs; D046912:Multiprotein Complexes; C486559:NCOA6 protein, human; D056918:Nuclear Receptor Coactivators; D010448:Peptide Initiation Factors; D043244:Ribonuclease III