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Defining the RNA interactome by total RNA‐associated protein purification
The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452921/ https://www.ncbi.nlm.nih.gov/pubmed/30962360 http://dx.doi.org/10.15252/msb.20188689 |
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author | Shchepachev, Vadim Bresson, Stefan Spanos, Christos Petfalski, Elisabeth Fischer, Lutz Rappsilber, Juri Tollervey, David |
author_facet | Shchepachev, Vadim Bresson, Stefan Spanos, Christos Petfalski, Elisabeth Fischer, Lutz Rappsilber, Juri Tollervey, David |
author_sort | Shchepachev, Vadim |
collection | PubMed |
description | The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA–protein complexes using silica beads. In a variant approach (PAR‐TRAPP), RNAs were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR‐TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR‐TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA–peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization. |
format | Online Article Text |
id | pubmed-6452921 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-64529212019-04-17 Defining the RNA interactome by total RNA‐associated protein purification Shchepachev, Vadim Bresson, Stefan Spanos, Christos Petfalski, Elisabeth Fischer, Lutz Rappsilber, Juri Tollervey, David Mol Syst Biol Methods The RNA binding proteome (RBPome) was previously investigated using UV crosslinking and purification of poly(A)‐associated proteins. However, most cellular transcripts are not polyadenylated. We therefore developed total RNA‐associated protein purification (TRAPP) based on 254 nm UV crosslinking and purification of all RNA–protein complexes using silica beads. In a variant approach (PAR‐TRAPP), RNAs were labelled with 4‐thiouracil prior to 350 nm crosslinking. PAR‐TRAPP in yeast identified hundreds of RNA binding proteins, strongly enriched for canonical RBPs. In comparison, TRAPP identified many more proteins not expected to bind RNA, and this correlated strongly with protein abundance. Comparing TRAPP in yeast and E. coli showed apparent conservation of RNA binding by metabolic enzymes. Illustrating the value of total RBP purification, we discovered that the glycolytic enzyme enolase interacts with tRNAs. Exploiting PAR‐TRAPP to determine the effects of brief exposure to weak acid stress revealed specific changes in late 60S ribosome biogenesis. Furthermore, we identified the precise sites of crosslinking for hundreds of RNA–peptide conjugates, using iTRAPP, providing insights into potential regulation. We conclude that TRAPP is a widely applicable tool for RBPome characterization. John Wiley and Sons Inc. 2019-04-08 /pmc/articles/PMC6452921/ /pubmed/30962360 http://dx.doi.org/10.15252/msb.20188689 Text en © 2019 The Authors. Published under the terms of the CC BY 4.0 license This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methods Shchepachev, Vadim Bresson, Stefan Spanos, Christos Petfalski, Elisabeth Fischer, Lutz Rappsilber, Juri Tollervey, David Defining the RNA interactome by total RNA‐associated protein purification |
title | Defining the RNA interactome by total RNA‐associated protein purification |
title_full | Defining the RNA interactome by total RNA‐associated protein purification |
title_fullStr | Defining the RNA interactome by total RNA‐associated protein purification |
title_full_unstemmed | Defining the RNA interactome by total RNA‐associated protein purification |
title_short | Defining the RNA interactome by total RNA‐associated protein purification |
title_sort | defining the rna interactome by total rna‐associated protein purification |
topic | Methods |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452921/ https://www.ncbi.nlm.nih.gov/pubmed/30962360 http://dx.doi.org/10.15252/msb.20188689 |
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