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



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Kafasla, Panagiota 
Cherny, Dmitry 
Llorian, Miriam 
Curry, Stephen 


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.



Alternative Splicing, Binding Sites, Mutation, Polypyrimidine Tract-Binding Protein, Protein Binding, Protein Structure, Tertiary, RNA Precursors, RNA, Messenger, fas Receptor

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Nucleic Acids Res

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Oxford University Press (OUP)
Biotechnology and Biological Sciences Research Council (BB/H004203/1)
Wellcome Trust (092900/Z/10/Z)
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)