Publication Date:
2022-05-26
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.
Description:
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:
This research was supported in part by a Discovery Grant (# 227158) from Natural Sciences &
Engineering Research Council (NSERC) (to CHL), University of Northern British Columbia Research
Project Awards (to MB, GVR, KM and SM), and the French National Research Agency/Hong Kong
Research Grants Council Joint Research Scheme (# A-HKUST601/13) (to ALM). DTK was a recipient of
NSERC Undergraduate Student Research Awards and a BC Cancer Agency Summer Studentship.
Keywords:
CRD-BP
;
RNA binding proteins
;
Mutagenesis
;
KH domain
;
Zebrafish
;
Granule formation
;
Ribonucleoprotein
;
RNA-protein interaction
;
mRNA
;
Molecular biology
Repository Name:
Woods Hole Open Access Server
Type:
Preprint
Format:
application/pdf
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