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Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide
The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are re...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726818/ https://www.ncbi.nlm.nih.gov/pubmed/26808308 http://dx.doi.org/10.1371/journal.pone.0148131 |
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author | Inchaustegui Gil, Diana P. Clayton, Christine |
author_facet | Inchaustegui Gil, Diana P. Clayton, Christine |
author_sort | Inchaustegui Gil, Diana P. |
collection | PubMed |
description | The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA. |
format | Online Article Text |
id | pubmed-4726818 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-47268182016-02-03 Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide Inchaustegui Gil, Diana P. Clayton, Christine PLoS One Research Article The cytoplasmic fates of mRNAs are influenced by interactions between RNA-binding proteins and cis regulatory motifs. In the cytoplasm, mRNAs are present as messenger ribonucleoprotein particles, which include not only proteins that bind directly to the mRNA, but also additional proteins that are recruited via protein-protein interactions. Many labs have sought to purify such particles from cells, with limited success. We here describe a simple two-step procedure to purify actively translated mRNAs, with their associated proteins, from polysomes. We use a reporter mRNA that encodes a protein with three streptavidin binding peptides at the N-terminus. The polysomal reporter mRNA, with associated proteins, is purified via binding to a streptavidin matrix. The method takes four days, and can be applied in any cell that can be genetically manipulated. Using Trypanosoma brucei as a model system, we routinely purified 8% of the input reporter mRNA, with roughly 22-fold enrichment relative to un-tagged mRNAs, a final reporter-mRNA:total-mRNA ratio of about 1:10, and a protein purification factor of slightly over 1000-fold. Although the overall reporter mRNP composition is masked by the presence of proteins that are associated with many polysomal mRNAs, our method can be used to detect association of an RNA-binding protein that binds to specifically to a reporter mRNA. Public Library of Science 2016-01-25 /pmc/articles/PMC4726818/ /pubmed/26808308 http://dx.doi.org/10.1371/journal.pone.0148131 Text en © 2016 Inchaustegui Gil, Clayton http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Inchaustegui Gil, Diana P. Clayton, Christine Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide |
title | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide |
title_full | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide |
title_fullStr | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide |
title_full_unstemmed | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide |
title_short | Purification of Messenger Ribonucleoprotein Particles via a Tagged Nascent Polypeptide |
title_sort | purification of messenger ribonucleoprotein particles via a tagged nascent polypeptide |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726818/ https://www.ncbi.nlm.nih.gov/pubmed/26808308 http://dx.doi.org/10.1371/journal.pone.0148131 |
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