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Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes

Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which...

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Detalles Bibliográficos
Autores principales: Trahan, Christian, Oeffinger, Marlene
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2016
Materias:
RNA
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756821/
https://www.ncbi.nlm.nih.gov/pubmed/26657640
http://dx.doi.org/10.1093/nar/gkv1366
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author Trahan, Christian
Oeffinger, Marlene
author_facet Trahan, Christian
Oeffinger, Marlene
author_sort Trahan, Christian
collection PubMed
description Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes.
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spelling pubmed-47568212016-02-18 Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes Trahan, Christian Oeffinger, Marlene Nucleic Acids Res RNA Proteomic and RNomic approaches have identified many components of different ribonucleoprotein particles (RNPs), yet still little is known about the organization and protein proximities within these heterogeneous and highly dynamic complexes. Here we describe a targeted cross-linking approach, which combines cross-linking from a known anchor site with affinity purification and mass spectrometry (MS) to identify the changing vicinity interactomes along RNP maturation pathways. Our method confines the reaction radius of a heterobifunctional cross-linker to a specific interaction surface, increasing the probability to capture low abundance conformations and transient vicinal interactors too infrequent for identification by traditional cross-linking-MS approaches, and determine protein proximities within RNPs. Applying the method to two conserved RNA-associated complexes in Saccharomyces cerevisae, the mRNA export receptor Mex67:Mtr2 and the pre-ribosomal Nop7 subcomplex, we identified dynamic vicinal interactomes within those complexes and along their changing pathway milieu. Our results therefore show that this method provides a new tool to study the changing spatial organization of heterogeneous dynamic RNP complexes. Oxford University Press 2016-02-18 2015-12-10 /pmc/articles/PMC4756821/ /pubmed/26657640 http://dx.doi.org/10.1093/nar/gkv1366 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle RNA
Trahan, Christian
Oeffinger, Marlene
Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
title Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
title_full Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
title_fullStr Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
title_full_unstemmed Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
title_short Targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous RNP complexes
title_sort targeted cross-linking-mass spectrometry determines vicinal interactomes within heterogeneous rnp complexes
topic RNA
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4756821/
https://www.ncbi.nlm.nih.gov/pubmed/26657640
http://dx.doi.org/10.1093/nar/gkv1366
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