Investigation of the Role of Gle1 in mRNA Export

描述(申请人提供)：信使RNA转录本(MRNAs)从细胞核输出到细胞质是真核基因表达的固有特性，然而，其机制尚不清楚。最近，Wente实验室发现了保守的mRNA输出因子Gle1和核Poly(A)结合蛋白NaB2之间的物理相互作用。该建议的目标是以萌芽中的酿酒酵母为模型系统，将Gle1：NaB2复合体表征为mRNA输出的关键终端步骤。从这项提议中获得的结果将描绘出Gle1的第一个功能角色，并将是基因表达领域的一个深刻进步。在目标I中，将使用经典生物化学和尖端质谱学来确定酵母中Gle1：NaB2复合体的性质以及该复合体在突变背景下的稳定性。此外，我将使用酵母遗传学和细胞显微镜技术来表征任何新发现的、新的信使核糖核酸输出因子。在AIM II中，我将开发一个体外组装系统，以确定Gle1：NaB2复合体是否与RNA结合，以及如果添加Gle1Nab2复合体的其他成员而改变RNA结合亲和力，这将为AIM I提供强大的体外补充。

DESCRIPTION (provided by applicant): Export of messenger RNA transcripts (mRNAs) from the nucleus to the cytoplasm is an inherent property of eukaryotic gene expression, however, the mechanism is poorly understood. Recently, the Wente laboratory discovered a physical interaction between the conserved mRNA export factor, Gle1 and the nuclear poly (A)- binding protein, Nab2. The goal of this proposal is to characterize the Gle1: Nab2 complex as a critical, terminal step in mRNA export using the budding yeast S. cerevisiae as a model system. Results gained from this proposal will delineate the first functional role of Gle1 and will be a profound advancement for the field of gene expression. In aim I, will use classic biochemistry coupled with cutting-edge mass spectrometry to determine the nature of the Gle1: Nab2 complex in yeast as well as the stability of this complex in mutant backgrounds. Additionally, I will employ techniques in yeast genetics and cellular microscopy to characterize any newly identified, novel mRNA export factors. In aim II, I will develop an in vitro assembly system to determine if the Gle1: Nab2 complex associates with RNA and if RNA-binding affinity is altered by addition of other members of the Gle1Nab2 complex, this provides a powerful, in vitro complement to aim I.