The organization of RNA contacts by PTB for regulation of FAS splicing
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Authors
Kafasla, Panagiota
Cherny, Dmitry
Llorian, Miriam
Curry, Stephen
Jackson, Richard
Smith, WJ
Publication Date
2014-06-23Journal Title
Nucleic Acids Research
ISSN
0305-1048
Publisher
Oxford University Press
Language
English
Type
Article
Metadata
Show full item recordCitation
Mickleburgh, I., Kafasla, P., Cherny, D., Llorian, M., Curry, S., Jackson, R., & Smith, W. (2014). The organization of RNA contacts by PTB for regulation of FAS splicing. Nucleic Acids Research https://doi.org/10.1093/nar/gku519
Abstract
Post-transcriptional steps of gene expression are regulated by RNA binding proteins.
Major progress has been made in characterizing RNA-protein interactions, from high
resolution structures to transcriptome-wide profiling. Due to the inherent technical
challenges, less attention has been paid to the way in which proteins with multiple
RNA binding domains engage with target RNAs. We have investigated how the four
RNA Recognition Motif (RRM) domains of Polypyrimidine Tract Binding protein
(PTB), a major splicing regulator, interact with FAS pre-mRNA under conditions in
which PTB represses FAS exon 6 splicing. A combination of tethered hydroxyl
radical probing, targeted inactivation of individual RRMs and single molecule
analyses revealed an unequal division of labour between the four RRMs of PTB.
RNA binding by RRM4 is the most important for function despite the low intrinsic binding
specificity and the complete lack of effect of disrupting individual RRM4 contact
points on the RNA. The ordered RRM3-4 di-domain packing provides an extended
binding surface for RNA interacting at RRM4, via basic residues in the preceding
linker. Our results illustrate how multiple alternative low-specificity binding
configurations of RRM4 are consistent with repressor function as long as the overall
ribonucleprotein architecture provided by appropriate di-domain packing is
maintained.
Sponsorship
This work was funded by grant support from the BBSRC to CWJS and R.J. Jackson
(BB/H004203/1) and in part by Wellcome Trust grant 092900. DC was supported by a
Wellcome Trust Value In People award (088113/Z/08/Z)
Funder references
BBSRC (BB/H004203/1)
Wellcome Trust (092900/Z/10/Z)
Identifiers
External DOI: https://doi.org/10.1093/nar/gku519
This record's URL: https://www.repository.cam.ac.uk/handle/1810/245394
Rights
Attribution 2.0 UK: England & Wales, Creative Commons Attribution License
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/
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