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

BMC Molecular Biology

Yoav Arava¹  Lilach Pnueli¹  Avigail Atir-Lande¹  Nitzan Samra¹ 

[1] Department of Biology, Technion-Israel Institute of Technology, Haifa, 32000, Israel

关键词: Protein translation;    Yeast;    3′ UTR;    Yef3;    Elongation factor 3;    RNA-binding proteins;   

Others  :  1227871
DOI  :  10.1186/s12867-015-0045-5

 received in 2015-05-11, accepted in 2015-09-17,  发布年份 2015

【 摘 要 】

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.

【 授权许可】

   
2015 Samra et al.

【 图 表 】

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