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

我的实验室旨在了解细胞质中mRNAs的翻译与内质网中mRNAs翻译的差异。我们计划揭示mRNAs是如何在这两个池之间分配的，并确定它们是如何受到明显监管的。 大约30%的人类蛋白质编码基因编码细胞膜和分泌蛋白质。尽管mRNA的内质网靶向被认为是共翻译的，但在过去的十年里，我的实验室与其他团队一起，证明了第二个基于RNA的靶向系统也存在。特别是，我的实验室发现了一种名为P180的mRNA受体，它招募并维持内质网表面的mRNAs。 为了进一步了解内质网和胞浆中mRNA的分类，我们从这两个隔室中纯化了mRNAs，并用质谱仪分析了它们的蛋白质组成。有趣的是，我们发现了大量的RNA结合蛋白优先与ER结合的mRNAs结合，包括许多翻译启动因子(包括eIF3复合体)。有趣的是，我们还发现糖酵解酶几乎只与胞浆相关的mRNAs相关。 在这项资助中，我概述了我们将如何跟踪我们的质谱学数据，以便更好地了解mRNAs是如何分配到内质网和胞浆的。 1)确定mRNAs是如何通过P180依赖的途径锚定到ER上的。 只有一部分mRNAs利用P180依赖的内质网靶向途径。然而，由于P180可能以一种序列无关的方式识别mRNAs，因此必须涉及识别特定基序的额外蛋白质。此外，可能还存在其他的信使核糖核酸受体，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假定的辅助蛋白和替代受体。在初步数据中，我们表明在我们的质谱学数据中确定的蛋白质之一是依赖于P180的ER锚定mRNAs所必需的。 2)深入了解糖酵解酶与mRNAs的关系。 在这个目标中，我们将调查糖酵解酶是否阻止mRNAs靶向内质网，或者调节主要编码胞浆蛋白的mRNAs的翻译。然后，我们研究这些酶是否将翻译调节与细胞的代谢状态结合起来。 3)确定翻译启动因子在内质网中丰富的机制。 在这个目标中，我们将确定翻译启动因子是直接与内质网捆绑，还是优先与在内质网上翻译的mRNA结合。然后，我们将确定它们的分布是否会因细胞压力而改变。 通过进行这一研究计划，我们将深入了解内质网上的mRNAs是如何与它们的细胞质同行相区别地受到调控的。我们的发现将使我们更深入地理解分泌是如何在mRNA定位的水平上进行调节的。

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.