Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains
van Rensburg, Gerrit
King, Dustin T.
Miller, Andrew L.
Lee, Chow H.
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KeywordCRD-BP; RNA binding proteins; Mutagenesis; KH domain; Zebrafish; Granule formation; Ribonucleoprotein; RNA-protein interaction; mRNA; Molecular biology
The ability of its four heterogeneous nuclear ribonucleoprotein-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of Coding Region Determinant-Binding Protein (CRD-BP). However, the particular RNA binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved Glycine-X-X-Glycine (GXXG) motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA-binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP a well as in the future design of inhibitors against CRDBP function.
Author Posting. © The Author(s), 2014. This is the author's version of the work. It is posted here by permission of American Society for Biochemistry and Molecular Biology for personal use, not for redistribution. The definitive version was published in Journal of Biological Chemistry 290 (2015): 625-639, doi:10.1074/jbc.M114.614735.
Suggested CitationPreprint: Barnes, Mark, van Rensburg, Gerrit, Li, Wai-Ming, Mehmood, Kashif, Mackedenski, Sebastian, Chan, Ching-Man, King, Dustin T., Miller, Andrew L., Lee, Chow H., "Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains", 2014-11, https://doi.org/10.1074/jbc.M114.614735, https://hdl.handle.net/1912/7140
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