Dissecting the differences between mRNA translation on the ER and in the cytoplasm in human cells

我的实验室的目的是了解mRNA在胞质溶胶和ER上的翻译之间的差异。我们计划揭示mRNA是如何在这两个池之间分配的，并确定它们是如何被不同地调节的。 大约30%的人类蛋白质编码基因编码膜蛋白和分泌蛋白。虽然ER靶向mRNA被认为是共同翻译的性质，在过去的十年里，我的实验室，沿着与其他小组，已经证明了第二个RNA为基础的靶向系统也存在。特别是，我的实验室发现了一种mRNA受体p180，它可以招募并维持ER表面的mRNA。 为了进一步了解mRNA在ER和胞质溶胶中的分选，我们从这两个隔室中纯化mRNA，并通过质谱分析其蛋白质组分。有趣的是，我们发现了大量的RNA结合蛋白优先与ER结合的mRNA，包括许多翻译起始因子（包括eIF3复合物）。有趣的是，我们还发现糖酵解酶几乎只与细胞溶质相关的mRNA相关。 在本研究中，我概述了我们将如何跟踪我们的质谱数据，以便更好地了解mRNA是如何分配到ER和胞质溶胶中的。 1)确定mRNA如何通过p180依赖性途径锚定到ER。 只有一部分mRNA利用p180依赖性ER靶向通路。然而，由于p180可能以不依赖于序列的方式识别mRNA，因此必须涉及识别特定基序的其他蛋白质。此外，可能还存在其他mRNA受体，尽管它们的身份仍然是个谜。在这个目标中，我们概述了我们将如何识别这些推定的辅助蛋白和替代受体。在初步的数据中，我们表明，在我们的质谱数据中确定的蛋白质之一是必需的p180依赖ER锚定的mRNA。 2)深入了解糖酵解酶与mRNA的关联。 在这个目标中，我们将研究糖酵解酶是否阻止mRNA靶向ER，或调节主要编码胞质蛋白的mRNA的翻译。然后，我们研究这些酶是否与细胞的代谢状态的翻译调控夫妇。 3)确定翻译起始因子在内质网中富集的机制。 在这个目标中，我们将确定翻译起始因子是否直接拴在ER或优先结合到ER上翻译的mRNA。然后我们将确定它们的分布是否被细胞应激改变。 通过进行这项研究计划，我们将深入了解ER上的mRNA是如何与其胞质对应物不同地调节的。我们的研究结果将提供一个更深入的了解分泌是如何在mRNA定位的水平上进行调节。

My lab aims to understand the differences between the translation of mRNAs in the cytosol and on the ER. We plan to uncover how mRNAs are partitioned between these two pools and to determine how they are distinctly regulated. Approximately 30% of all human protein-coding genes, code for membrane and secretory proteins. Although ER-targeting of mRNA is thought to be co-translational in nature, over the past decade, my lab, along with other groups, have demonstrated that a second RNA-based targeting system also exists. In particular my lab discovered an mRNA receptor, p180, which recruits and then maintains mRNAs on the surface of the ER. To gain further insight into the sorting of mRNA to the ER and the the cytosol, we purified mRNAs from these two compartments and analyzed their protein constituents by mass spectrometry. Interestingly, we identified a large number of RNA-binding proteins associate preferentially with ER-bound mRNAs including many translation initiation factors (including the eIF3 complex). Intriguingly, we also discovered that glycolysis enzymes are associated almost exclusively with cytosolic-associated mRNAs. In this grant I outline how we will follow up on our mass spectrometry data in order to better understand how mRNAs are partitioned to the ER and cytosol. 1) Determining how mRNAs are anchored to the ER by the p180-dependent pathway. Only a subset of mRNAs utilize the p180-dependent ER-targeting pathway. However, since p180 likely recognizes mRNAs in a sequence-independent manner, additional proteins which recognize particular motifs must be involved. In addition, other mRNA receptors likely exist, although their identity remains mysterious. In this aim we outline how we will identify these putative accessory proteins and alternative receptors. In preliminary data we show that one of the proteins identified in our mass spec data is required for p180-dependent ER-anchoring of mRNAs. 2) Gaining insight into the association of glycolysis enzymes with mRNAs. In this aim we will investigate whether glycolysis enzymes are either preventing mRNAs from targeting to the ER, or regulating the translation of mRNAs that encode predominantly cytosolic proteins. We then investigate whether these enzymes couple translation regulation with the metabolic status of the cell. 3) Determining the mechanism by which translation initiation factors are enriched in the ER. In this aim we will determine whether translation initiation factors are directly tethered to the ER or are preferentially bound to mRNAs that are translated on the ER. We then will determine whether their distribution is altered by cellular stress. By pursuing this research program we will gain insight into how mRNAs on the ER are regulated distinctly from their cytosolic counterparts. Our findings will provide a deeper understanding into how secretion is regulated at the level of mRNA localization.