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Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins

We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and i...

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Autores principales: Klein, Cornelia, Terrao, Monica, Inchaustegui Gil, Diana, Clayton, Christine
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545788/
https://www.ncbi.nlm.nih.gov/pubmed/26287607
http://dx.doi.org/10.1371/journal.pone.0135973
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author Klein, Cornelia
Terrao, Monica
Inchaustegui Gil, Diana
Clayton, Christine
author_facet Klein, Cornelia
Terrao, Monica
Inchaustegui Gil, Diana
Clayton, Christine
author_sort Klein, Cornelia
collection PubMed
description We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.
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spelling pubmed-45457882015-09-01 Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins Klein, Cornelia Terrao, Monica Inchaustegui Gil, Diana Clayton, Christine PLoS One Research Article We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites. Public Library of Science 2015-08-19 /pmc/articles/PMC4545788/ /pubmed/26287607 http://dx.doi.org/10.1371/journal.pone.0135973 Text en © 2015 Klein et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Klein, Cornelia
Terrao, Monica
Inchaustegui Gil, Diana
Clayton, Christine
Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins
title Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins
title_full Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins
title_fullStr Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins
title_full_unstemmed Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins
title_short Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins
title_sort polysomes of trypanosoma brucei: association with initiation factors and rna-binding proteins
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545788/
https://www.ncbi.nlm.nih.gov/pubmed/26287607
http://dx.doi.org/10.1371/journal.pone.0135973
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