Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA.
Mammalian Dicer interacts with double-stranded RNA-binding protein TRBP or PACT to mediate RNA interference and micro-RNA processing. TRBP and PACT are structurally related but exert opposite regulatory activities on PKR. It is not understood whether TRBP and PACT are simultaneously required for Dicer. Here we show that TRBP directly interacts with PACT in vitro and in mammalian cells. TRBP and PACT form a triple complex with Dicer and facilitate the production of small interfering RNA (siRNA) by Dicer. Knockdown of both TRBP and PACT in cultured cells leads to significant inhibition of gene silencing mediated by short hairpin RNA but not by siRNA, suggesting that TRBP and PACT function primarily at the step of siRNA production. Taken together, these findings indicate that human TRBP and PACT directly interact with each other and associate with Dicer to stimulate the cleavage of double-stranded or short hairpin RNA to siRNA. Our work significantly alters the current model for the assembly and function of the Dicer-containing complex that generates siRNA and micro-RNA in human.
Kok
Kin Hang
KH
Department of Biochemistry, University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China.
Ng
Ming-Him James
MH
Ching
Yick-Pang
YP
Jin
Dong-Yan
DY
D000818:Animals; D031541:Fluorescence Resonance Energy Transfer; D020868:Gene Silencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D051379:Mice; D046912:Multiprotein Complexes; D056918:Nuclear Receptor Coactivators; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics / Q000378:metabolism*; D013737:Testis / Q000378:metabolism
D047908:Intracellular Signaling Peptides and Proteins; D046912:Multiprotein Complexes; C486559:NCOA6 protein, human; D056918:Nuclear Receptor Coactivators; C104647:PRKRA protein, human; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D012313:RNA; D043244:Ribonuclease III