Investigation of the Role of Gle1 in mRNA Export

信使RNA转录本(MRNAs)从细胞核输出到细胞质是 然而，对于真核基因表达的机制却知之甚少。最近，温特实验室 发现保守的mRNA输出因子Gle1与核多聚(A)之间的物理相互作用- 结合蛋白，NaB2。这项提议的目标是将Gle1：NaB2复合体描述为关键的、 以发芽酵母酿酒酵母为模型系统，实现了信使核糖核酸输出的末端步骤。取得的成果 这项提案将勾勒出Gle1的第一个职能角色，并将是对 基因表达领域。在AIM I中，将使用经典的生物化学和尖端的质谱学 确定酵母中Gle1：NaB2复合体的性质以及该复合体在突变体中的稳定性 背景。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来 确定Gle1：NaB2复合体是否与RNA结合，以及RNA结合亲和力是否因添加 Gle1Nab2复合体的其他成员，这为AIM I提供了一个强大的体外补充。

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 stabiility 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.