Mechanism and regulation of mRNA export

mRNA输出的机制和调控

• 批准号: 7038607
• 负责人: RAVI DHAR
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7038607
• 关键词: Schizosaccharomyces pombe; biological models; eukaryote; fungal genetics; fungal proteins; immunoprecipitation; intracellular transport; messenger RNA; model design /development; nuclear membrane; nuclear transport; pore forming protein; protein protein interaction; protein purification; protein transport; transport proteins; tumor suppressor proteins

Nuclear export of mRNA is a fundamentally important process that is conserved in evolution. Mature mRNA is exported through the nuclear pores bound to proteins as an RNP-particle. In the cytoplasm, the RNP-complex is disassembled, the mRNA is released for translation and the proteins involved in the export of mRNA are returned to the nucleus utilizing import pathways so that they can participate in another round of mRNA export. We have used fission yeast Schizosaccharomyces pombe (S. pombe) as a model system to study the mechanism of how eukaryotic cells export their mRNA out of the nucleus. A variety of experiments suggest that both yeast Mex67/metazoan Tap and Rae1 are involved in the export of mRNA at steps after the maturation of mRNA, including those that link the mRNA-protein complex to the nuclear pore. The identity of many of the soluble factors, including some protein-protein interactions of the Mex67 mediated mRNA export pathway are known. Our studies are designed to identify protein-protein interactions of the Rae1p mediated mRNA export pathway. We have found that the S. pombe homologs of S. cerevisiae Yra1p, Sub2p, Sac3p that function with Mex67/Mtr2 proteins in S. cerevisiae are components of the Rae1p-mediated mRNA export pathway in S. pombe. In S. pombe they are known as Mlo3p, Uap56p and Sac3p respectively. Additionally, Dss1p, the S. pombe homolog of human breast cancer tumor suppressor (BRAC2) cofactor DSS1, is a novel mRNA export factor that functions with Rae1p. By using purified proteins we found that Dss1p, Mlo3p, Uap56p and Rae1p interact in vitro. Importantly, Dss1p and Uap56p appear to act as linkers between Mlo3p and Rae1p. Tap-tagged Dss1 could immunoprecipitate the interacting proteins in vivo confirming the in vitro data. Based on these experiments we have developed a mechanistic model for the essential Rae1p-mediated mRNA export route in S. pombe. In essence, in this model Dss1p and Uap56p could link mRNA to the nuclear pores by bridging Mlo3p to Rae1p.

mRNA的核出口是一个从进化中保守的根本重要过程。成熟的mRNA通过与蛋白质作为RNP粒子结合的核孔输出。在细胞质中，RNP复合物被拆卸，mRNA被释放进行翻译，并且涉及MRNA导出的蛋白质使用进口途径返回到原子核中，以便它们可以参与另一轮mRNA导出。我们已经使用裂变酵母菌型糖菌（S. pombe）作为模型系统，以研究真核细胞如何将其mRNA从细胞核中导出的机制。各种实验表明，在mRNA成熟后，酵母菌MEX67/MEXAXOAN TAP和RAE1都参与了MRNA的出口，包括将mRNA-蛋白质复合物与核孔联系起来的mRNA。许多可溶性因子的身份，包括MEX67介导的mRNA输出途径的某些蛋白质 - 蛋白质相互作用。我们的研究旨在鉴定RAE1P介导的mRNA输出途径的蛋白质蛋白质相互作用。我们发现，酿酒酵母YRA1P，SUB2P，SAC3P的S. pombe同源物在酿酒酵母中与MEX67/MTR2蛋白一起发挥作用，是S. pombe中RAE1P介导的mRNA Export途径的组件。在S. pombe中，它们分别称为MLO3P，UAP56P和SAC3P。此外，DSS1P是人类乳腺癌肿瘤抑制剂（BRAC2）辅因子DSS1的POMBE同源物，是一种具有RAE1p功能的新型mRNA输出因子。通过使用纯化的蛋白质，我们发现DSS1P，MLO3P，UAP56P和RAE1P在体外相互作用。重要的是，DSS1P和UAP56P似乎是MLO3P和RAE1P之间的接头。 TAP标记的DSS1可以在体内免疫沉淀的相互作用蛋白，从而确认体外数据。基于这些实验，我们为S. pombe中的必需RAE1P介导的mRNA导出途径开发了一种机械模型。本质上，在此模型中，DSS1P和UAP56P可以通过将MLO3P桥接到RAE1P将mRNA与核孔联系起来。