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The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner

BACKGROUND: mRNA binding proteins (RBPs) constitute 10–15 % of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature its interaction with RBPs, identification of novel RBPs is a significant challe...

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Autores principales: Samra, Nitzan, Atir-Lande, Avigail, Pnueli, Lilach, Arava, Yoav
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582935/
https://www.ncbi.nlm.nih.gov/pubmed/26404137
http://dx.doi.org/10.1186/s12867-015-0045-5
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author Samra, Nitzan
Atir-Lande, Avigail
Pnueli, Lilach
Arava, Yoav
author_facet Samra, Nitzan
Atir-Lande, Avigail
Pnueli, Lilach
Arava, Yoav
author_sort Samra, Nitzan
collection PubMed
description BACKGROUND: mRNA binding proteins (RBPs) constitute 10–15 % of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature its interaction with RBPs, identification of novel RBPs is a significant challenge. Recently, a novel methodology for RBP purification and identification (termed RaPID) was presented, which allows high affinity purification of RBPs while associated with mRNA in vivo. RESULTS: We performed a RaPID screen for proteins that interact with PMP1 mRNA in order to identify novel mRNA binding proteins. PMP1 mRNA was tagged in its 3′ UTR with multiple MS2 loops and co-expressed with MS2-binding protein fused to streptavidin binding protein (SBP). RNA–protein complexes were cross-linked in vivo and isolated through streptavidin beads. The eluted proteins were subjected to mass spectroscopy analysis. The screen identified many proteins, about half of them were previously shown to bind RNA. We focused on eEF3 (YEF3), an essential translation elongation factor that interacts with ribosomes. Purification of TAP-tagged Yef3 with its associated RNAs confirmed that the native PMP1 transcript is associated with it. Intriguingly, high association with Yef3-TAP was observed when purification was performed in the presence of EDTA, and with PMP1 that contains stop codons immediately downstream to the initiation codon. Furthermore, high association was observed with a transcript containing only the 3′ UTR of PMP1. Complementary, RaPID isolation of MS2-tagged 3′ UTRs with their associated proteins revealed that Yef3 can efficiently interact with these regions. CONCLUSIONS: This study identifies many novel proteins that interact with PMP1 mRNA. Importantly, the elongation factor Yef3 was found to interact with mRNA in non-coding regions and in a translation independent manner. These results suggest an additional, non-elongation function for this factor. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12867-015-0045-5) contains supplementary material, which is available to authorized users.
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spelling pubmed-45829352015-09-26 The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner Samra, Nitzan Atir-Lande, Avigail Pnueli, Lilach Arava, Yoav BMC Mol Biol Research Article BACKGROUND: mRNA binding proteins (RBPs) constitute 10–15 % of the eukaryotic proteome and play important part in post-transcriptional regulation of gene expression. Due to the instability of RNA and the transient nature its interaction with RBPs, identification of novel RBPs is a significant challenge. Recently, a novel methodology for RBP purification and identification (termed RaPID) was presented, which allows high affinity purification of RBPs while associated with mRNA in vivo. RESULTS: We performed a RaPID screen for proteins that interact with PMP1 mRNA in order to identify novel mRNA binding proteins. PMP1 mRNA was tagged in its 3′ UTR with multiple MS2 loops and co-expressed with MS2-binding protein fused to streptavidin binding protein (SBP). RNA–protein complexes were cross-linked in vivo and isolated through streptavidin beads. The eluted proteins were subjected to mass spectroscopy analysis. The screen identified many proteins, about half of them were previously shown to bind RNA. We focused on eEF3 (YEF3), an essential translation elongation factor that interacts with ribosomes. Purification of TAP-tagged Yef3 with its associated RNAs confirmed that the native PMP1 transcript is associated with it. Intriguingly, high association with Yef3-TAP was observed when purification was performed in the presence of EDTA, and with PMP1 that contains stop codons immediately downstream to the initiation codon. Furthermore, high association was observed with a transcript containing only the 3′ UTR of PMP1. Complementary, RaPID isolation of MS2-tagged 3′ UTRs with their associated proteins revealed that Yef3 can efficiently interact with these regions. CONCLUSIONS: This study identifies many novel proteins that interact with PMP1 mRNA. Importantly, the elongation factor Yef3 was found to interact with mRNA in non-coding regions and in a translation independent manner. These results suggest an additional, non-elongation function for this factor. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12867-015-0045-5) contains supplementary material, which is available to authorized users. BioMed Central 2015-09-24 /pmc/articles/PMC4582935/ /pubmed/26404137 http://dx.doi.org/10.1186/s12867-015-0045-5 Text en © Samra et al. 2015 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Samra, Nitzan
Atir-Lande, Avigail
Pnueli, Lilach
Arava, Yoav
The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
title The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
title_full The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
title_fullStr The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
title_full_unstemmed The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
title_short The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
title_sort elongation factor eef3 (yef3) interacts with mrna in a translation independent manner
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4582935/
https://www.ncbi.nlm.nih.gov/pubmed/26404137
http://dx.doi.org/10.1186/s12867-015-0045-5
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