RNA helicase A interacts with RISC in human cells and functions in RISC loading.
RNA interference is a conserved pathway of sequence-specific gene silencing that depends on small guide RNAs and the action of proteins assembled in the RNA-induced silencing complex (RISC). Minimally, the action of RISC requires the endonucleolytic slicer activity of Argonaute2 (Ago2) directed to RNA targets whose sequences are complementary to RISC-incorporated small RNA. To identify RISC components in human cells, we developed an affinity-purification strategy to isolate siRNA-programmed RISC. Here we report the identification of RNA helicase A (RHA) as a human RISC-associated factor. We show that RHA interacts in human cells with siRNA, Ago2, TRBP, and Dicer and functions in the RNAi pathway. In RHA-depleted cells, RNAi was reduced as a consequence of decreased intracellular concentration of active RISC assembled with the guide-strand RNA and Ago2. Our results identify RHA as a RISC component and demonstrate that RHA functions in RISC as an siRNA-loading factor.
Robb
G Brett
GB
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Rana
Tariq M
TM
D060565:Argonaute Proteins; D002268:Carboxypeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism; D020868:Gene Silencing; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D002268:Carboxypeptidases; C436327:SCPEP1 protein, human; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases