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The organization of RNA contacts by PTB for regulation of FAS splicing
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...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2014
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117754/ https://www.ncbi.nlm.nih.gov/pubmed/24957602 http://dx.doi.org/10.1093/nar/gku519 |
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author | Mickleburgh, Ian Kafasla, Panagiota Cherny, Dmitry Llorian, Miriam Curry, Stephen Jackson, Richard J. Smith, Christopher W.J. |
author_facet | Mickleburgh, Ian Kafasla, Panagiota Cherny, Dmitry Llorian, Miriam Curry, Stephen Jackson, Richard J. Smith, Christopher W.J. |
author_sort | Mickleburgh, Ian |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-4117754 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-41177542014-08-15 The organization of RNA contacts by PTB for regulation of FAS splicing Mickleburgh, Ian Kafasla, Panagiota Cherny, Dmitry Llorian, Miriam Curry, Stephen Jackson, Richard J. Smith, Christopher W.J. Nucleic Acids Res RNA 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. Oxford University Press 2014-09-01 2014-06-21 /pmc/articles/PMC4117754/ /pubmed/24957602 http://dx.doi.org/10.1093/nar/gku519 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/3.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | RNA Mickleburgh, Ian Kafasla, Panagiota Cherny, Dmitry Llorian, Miriam Curry, Stephen Jackson, Richard J. Smith, Christopher W.J. The organization of RNA contacts by PTB for regulation of FAS splicing |
title | The organization of RNA contacts by PTB for regulation of FAS splicing |
title_full | The organization of RNA contacts by PTB for regulation of FAS splicing |
title_fullStr | The organization of RNA contacts by PTB for regulation of FAS splicing |
title_full_unstemmed | The organization of RNA contacts by PTB for regulation of FAS splicing |
title_short | The organization of RNA contacts by PTB for regulation of FAS splicing |
title_sort | organization of rna contacts by ptb for regulation of fas splicing |
topic | RNA |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4117754/ https://www.ncbi.nlm.nih.gov/pubmed/24957602 http://dx.doi.org/10.1093/nar/gku519 |
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