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首页> 外文期刊>BMC Molecular Biology >The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner
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The elongation factor eEF3 (Yef3) interacts with mRNA in a translation independent manner

机译：延伸因子eEF3（Yef3）以​​不依赖翻译的方式与mRNA相互作用

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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. 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. 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.
机译：mRNA结合蛋白（RBPs）构成真核蛋白质组的10-15％，并在基因表达的转录后调控中发挥重要作用。由于RNA的不稳定性及其与RBP相互作用的瞬时性质，鉴定新的RBP是一项重大挑战。最近，提出了一种用于RBP纯化和鉴定的新方法（称为RaPID），它可以在体内与mRNA结合的同时对RBP进行高亲和力纯化。我们进行了与PMP1 mRNA相互作用的蛋白的RaPID筛选，以鉴定新颖的mRNA结合蛋白。 PMP1 mRNA在其3'UTR中带有多个MS2环标记，并与融合至链霉亲和素结合蛋白（SBP）的MS2结合蛋白共表达。 RNA-蛋白复合物在体内经过交联，并通过链霉亲和素珠分离。对洗脱的蛋白质进行质谱分析。筛选过程中鉴定出许多蛋白质，其中大约一半以前显示与RNA结合。我们专注于eEF3（YEF3），这是一种与核糖体相互作用的必不可少的翻译延伸因子。 TAP标签的Yef3及其相关RNA的纯化证实了天然PMP1转录物与其相关。有趣的是，在EDTA存在下进行纯化时，观察到与Yef3-TAP的高度缔合，而在起始密码子的紧下游则发现含有终止密码子的PMP1。此外，观察到与仅包含PMP1的3'UTR的转录物高度相关。 MS2标签的3'UTR及其相关蛋白的互补，RaPID分离显示，Yef3可与这些区域有效相互作用。这项研究确定了许多与PMP1 mRNA相互作用的新型蛋白质。重要的是，发现延伸因子Yef3与非编码区中的mRNA相互作用并且以不依赖翻译的方式相互作用。这些结果表明该因子具有附加的非伸长功能。

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