Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9).
Intensive research interest has focused on small RNA-processing machinery and the RNA-induced silencing complex (RISC), key cellular machines in RNAi pathways. However, the structural mechanism regarding RISC assembly, the primary step linking small RNA processing and RNA-mediated gene silencing, is largely unknown. Human RNA helicase A (DHX9) was reported to function as an RISC-loading factor, and such function is mediated mainly by its dsRNA-binding domains (dsRBDs). Here, we report the crystal structures of human RNA helicase A (RHA) dsRBD1 and dsRBD2 domains in complex with dsRNAs, respectively. Structural analysis not only reveals higher siRNA duplex-binding affinity displayed by dsRBD1, but also identifies a crystallographic dsRBD1 pair of physiological significance in cooperatively recognizing dsRNAs. Structural observations are further validated by isothermal titration calorimetric (ITC) assay. Moreover, co-immunoprecipitation (co-IP) assay coupled with mutagenesis demonstrated that both dsRBDs are required for RISC association, and such association is mediated by dsRNA. Hence, our structural and functional efforts have revealed a potential working model for siRNA recognition by RHA tandem dsRBDs, and together they provide direct structural insights into RISC assembly facilitated by RHA.
Department of Biological Sciences and Centre for Bioimaging Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Singapore.
Qinqin
Q
Fu
Y Adam
YA
Yuan
D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000737:chemistry*; D034741:RNA, Small Interfering / Q000737:chemistry; D034743:RNA-Induced Silencing Complex / Q000737:chemistry*; D040681:Structural Homology, Protein
D009363:Neoplasm Proteins; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; C098487:DHX9 protein, human; D053487:DEAD-box RNA Helicases