Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains
Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains
Date
2014-11
Authors
Barnes, Mark
van Rensburg, Gerrit
Li, Wai-Ming
Mehmood, Kashif
Mackedenski, Sebastian
Chan, Ching-Man
King, Dustin T.
Miller, Andrew L.
Lee, Chow H.
van Rensburg, Gerrit
Li, Wai-Ming
Mehmood, Kashif
Mackedenski, Sebastian
Chan, Ching-Man
King, Dustin T.
Miller, Andrew L.
Lee, Chow H.
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Keywords
CRD-BP
RNA binding proteins
Mutagenesis
KH domain
Zebrafish
Granule formation
Ribonucleoprotein
RNA-protein interaction
mRNA
Molecular biology
RNA binding proteins
Mutagenesis
KH domain
Zebrafish
Granule formation
Ribonucleoprotein
RNA-protein interaction
mRNA
Molecular biology
Abstract
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.
Description
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.