Molecular insights into the coding region determinant-binding protein-RNA interaction through site-directed mutagenesis in the heterogeneous nuclear ribonucleoprotein-K-homology domains.
The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the 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 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 the 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 as well as in the future design of inhibitors against CRD-BP function.
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.
Mark
M
Barnes
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.
Gerrit
G
van Rensburg
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.
Wai-Ming
WM
Li
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.
Kashif
K
Mehmood
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.
Sebastian
S
Mackedenski
the Division of Life Science and The Key State Laboratory for Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, and.
Ching-Man
CM
Chan
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada.
Dustin T
DT
King
the Division of Life Science and The Key State Laboratory for Molecular Neuroscience, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, and the Marine Biological Laboratory, Woods Hole, Massachusetts 02543.
Andrew L
AL
Miller
From the Chemistry Program, University of Northern British Columbia, Prince George, British Columbia V2N 4Z9, Canada, chow.lee@unbc.ca.
Chow H
CH
Lee
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004625:Embryo, Nonmammalian; D015870:Gene Expression; D005998:Glycine / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D018960:Hyaluronan Receptors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D016366:Open Reading Frames; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D020598:Proto-Oncogene Proteins c-myb / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012334:RNA, Neoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology / Q000235:genetics* / Q000378:metabolism
C497483:CD44 protein, human; D018960:Hyaluronan Receptors; C406144:IGF2BP1 protein, human; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D001224:Aspartic Acid; D005998:Glycine
RNA; RNA Metabolism; RNA Processing; RNA Turnover; RNA-Protein Interaction; RNA-binding Protein