Complexity and function of messenger ribonucleoprotein particles

细胞不仅通过控制转录，而且通过调节其mRNAs的定位、稳定性和翻译率来决定其遗传程序的最终蛋白质输出。我们的实验室正在研究在真核生物进化的两个广泛分离的分支：锥体和哺乳动物中，在转录后水平控制基因表达的机制。最终，任何给定的mRNA的命运都取决于所有相关的RNA结合蛋白(RBPs)的集合，即信使核糖核蛋白颗粒(MRNP)。然而，令人惊讶的是，人们对mRNPs的组成知之甚少。在这项拨款申请中，我们希望通过纯化多肽新生链并将RNA亲和性标签引入报告mRNAs来表征锥虫和哺乳动物细胞系中的mRNPs。我们的具体目标是1)鉴定编码可变表面糖蛋白的mRNP，可变表面糖蛋白是血流锥虫中含量最丰富的蛋白质；2)比较锥虫和哺乳动物产生α-微管蛋白的mRNP，微管蛋白是真核生物进化中高度丰富和保守的蛋白质；以及3)鉴定与我们在编码小鼠和人类肿瘤坏死因子α的mRNAs中发现的一种新的RNA衰变元件相关的限制性商业惯例。这些研究不仅将提高我们对真核mRNPs的基本理解，而且还将为解决RNA生物学领域的许多问题提供有用的方法。

Cells determine the final protein output of their genetic program not only by controlling transcription, but also by regulating the localization, stabilities and translation rates of their mRNAs. Our laboratories are investigating mechanisms that control gene expression at the post-transcriptional level in two widely separated branches of eukaryotic evolution: trypanosomes and mammals. Ultimately, the fate of any given mRNA is determined by the ensemble of all associated RNA-binding proteins (RBPs), i.e. the messenger ribonucleoprotein particle (mRNP). Surprisingly little, however, is known about the composition of mRNPs. In this grant application, we want to characterize mRNPs from trypanosomes and mammalian cell lines by purification of polypeptide nascent chains, and by introducing RNA affinity tags into reporter mRNAs. Our specific goals are 1) to characterize the mRNP encoding the Variable Surface Glycoprotein, the most abundant protein of bloodstream trypanosomes; 2) to compare the trypanosome and mammalian mRNPs producing α-tubulin, a highly abundant and conserved protein throughout eukaryotic evolution; and 3) to identify RBPs associated with a novel RNA decay element that we found in the mRNAs encoding mouse and human tumor necrosis factor-α. These studies will not only improve our basic understanding of eukaryotic mRNPs, but also provide methods that are useful in addressing many questions in the field of RNA biology.