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The organization of RNA contacts by PTB for regulation of FAS splicing

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journal contribution
posted on 27.04.2015, 15:00 by I. Mickleburgh, P. Kafasla, Dmitry Cherny, M. Llorian, S. Curry, R. J. Jackson, C. W. Smith
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 (PTB) protein, 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 ribonucleoprotein architecture provided by appropriate di-domain packing is maintained.

Funding

BBSRC [BB/H004203/1 to C.W.J.S. and R.J.J.]; Wellcome Trust [092900]. Wellcome Trust Value In People award [088113/Z/08/Z to D.C.]. Source of open access funding: the RCUK block grant to the University of Cambridge.

History

Citation

Nucleic Acids Research, 2014, 42 (13), pp. 8605-8620

Author affiliation

/Organisation/COLLEGE OF MEDICINE, BIOLOGICAL SCIENCES AND PSYCHOLOGY/School of Biological Sciences/Department of Biochemistry

Version

VoR (Version of Record)

Published in

Nucleic Acids Research

Publisher

Oxford University Press (OUP)

issn

0305-1048

eissn

1362-4962

Copyright date

2014

Available date

27/04/2015

Publisher version

http://nar.oxfordjournals.org/content/42/13/8605

Notes

PMCID: PMC4117754

Language

en