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...
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Oxford University Press
2013
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| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597700/ |
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pubmed-35977002013-03-15 Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) Fu, Qinqin Yuan, Y. Adam Structural Biology 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. Oxford University Press 2013-03 2013-01-29 /pmc/articles/PMC3597700/ /pubmed/23361462 http://dx.doi.org/10.1093/nar/gkt042 Text en © The Author(s) 2013. Published by Oxford University Press. http://creativecommons.org/licenses/by-nc/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. |
| repository_type |
Open Access Journal |
| institution_category |
Foreign Institution |
| institution |
US National Center for Biotechnology Information |
| building |
NCBI PubMed |
| collection |
Online Access |
| language |
English |
| format |
Online |
| author |
Fu, Qinqin Yuan, Y. Adam |
| spellingShingle |
Fu, Qinqin Yuan, Y. Adam Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) |
| author_facet |
Fu, Qinqin Yuan, Y. Adam |
| author_sort |
Fu, Qinqin |
| title |
Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) |
| title_short |
Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) |
| title_full |
Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) |
| title_fullStr |
Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) |
| title_full_unstemmed |
Structural insights into RISC assembly facilitated by dsRNA-binding domains of human RNA helicase A (DHX9) |
| title_sort |
structural insights into risc assembly facilitated by dsrna-binding domains of human rna helicase a (dhx9) |
| description |
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. |
| publisher |
Oxford University Press |
| publishDate |
2013 |
| url |
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3597700/ |
| _version_ |
1611962185460744192 |