Nonsense-mediated mRNA decay: Pioneer round of translation

描述（由申请人提供）：我们的目标是继续我们的研究先驱翻译启动复杂和翻译的先驱轮。无义介导的mRNA衰变（NMD）是一种重要的质量控制机制，其消除具有产生可能对细胞有害的截短蛋白质的潜力的转录物。我们已经发现，哺乳动物细胞中的NMD通常发生在翻译的先锋轮期间，当翻译终止于剪接产生的外显子-外显子连接点上游约50 - 55 nt时。根据定义，这一轮翻译利用由帽结合蛋白异二聚体CBP 80/20结合的mRNA。外显子-外显子连接在NMD中的作用反映了包括Upf NMD因子的蛋白质的外显子连接复合物（EJC）的剪接后沉积。我们已经发现EJC代表剪接的CBP 80/20结合的mRNA，但不是其重塑产物eIF 4 E结合的mRNA。到目前为止，我们已经表明，先锋翻译起始复合物在功能上不同于稳态，但在结构上与稳态重叠，即，eIF 4 E结合的翻译起始复合物。我们已经查明了国家导弹防御系统所涉及的一些破坏性活动。我们也开始解开为什么一些mRNA受到核相关的NMD，而其他mRNA受到细胞质NMD。目的一：进一步研究翻译前和翻译过程中先驱翻译起始复合物的结构，并对我们已经发现的CBP 80-Upf 1、CBP 80-SMG 1、CBP 80-eIF 4GI、Upf 1 -eRF 1和Upf 1 -eRF 3的相互作用结构域进行表征。在目标2中，我们将确定为什么一些mRNA针对核相关NMD，而其他mRNA针对细胞质NMD，并且与Rob Singer的实验室合作，我们将使用单个RNA分子的荧光原位杂交定位核相关NMD的细胞位点。在目标3中，我们将巩固与Ben Blencowe实验室合作获得的数据，这些数据表明不同EJC之间存在功能差异，或者位于无义密码子下游的任何EJC是否可以触发NMD。将利用这些差异来确定NMD是否仅由最3 '端的EJC触发，或者位于无义密码子下游超过50-55个核苷酸的任何EJC是否可以触发NMD。我们希望，对这笔赠款的重新支持将使我们能够继续在了解哺乳动物细胞中的NMD机制方面取得重大进展。

DESCRIPTION (provided by applicant): We aim to continue our studies of the pioneer translation initiation complex and the pioneer round of translation. Nonsense-mediated mRNA decay (NMD) is an important quality control mechanism that eliminates transcripts having the potential to generate truncated proteins that could be deleterious to cells. We have found that NMD in mammalian cells generally occurs when translation terminates more than -50- 55 nts upstream of a splicing-generated exon-exon junction during a pioneer round of translation. By definition, this round of translation utilizes mRNA that is bound by the cap binding protein heterodimer CBP80/20. The role of the exon-exon junction in NMD reflects the post-splicing deposition of an exon junction complex (EJC) of proteins that include the Upf NMD factors. We have found that EJCs typify spliced CBP80/20-bound mRNA but not its remodeled product, elF4E-bound mRNA. To date, we have shown that the pioneer translation initiation complex is functionally distinct from but structurally overlaps with the steady- state, i.e., elF4E-bound, translation initiation complex. We have identified degradative activities involved in NMD. We also began to unravel why some mRNAs are subject to nucleus-associated NMD whereas other mRNAs are subject to cytoplasmic NMD. In Aim 1, we will study further the structure of the pioneer translation initiation complex before and during translation, and we will characterize the interacting domains of CBP80-Upf 1, CBP80-SMG1, CBP80-elF4GI, Upf 1 -eRF1 and Upf 1 -eRF3 that we have shown exist. In Aim 2, we will determine why some mRNAs are targeted for nucleus-associated NMD and other mRNAs are targeted for cytoplasmic NMD and, in collaboration with Rob Singer's lab, we will localize the cellular site of nucleus-associated NMD using fluorescent in situ hybridization of single RNA molecules. In Aim 3, we will solidify data obtained in collaboration with Ben Blencowe's lab indicating that there are functional differences among different EJCs or if any EJC that resides sufficiently downstream of a nonsense codon can trigger NMD. These differences will be exploited to determine if NMD is triggered by only the 3'-most EJC or if any EJC that resides more than 50-55 nucleotides downstream of a nonsense codon can trigger NMD. We expect that renewed support of this grant will allow us to continue to make major advances in understanding the mechanism of NMD in mammalian cells.