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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...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2016
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Materias: | |
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. |
format | Online Article Text |
id | pubmed-4756821 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
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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