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 的核输出是进化中保守的一个极其重要的过程。成熟的 mRNA 通过与蛋白质结合的核孔作为 RNP 颗粒输出。在细胞质中，RNP复合物被分解，mRNA被释放进行翻译，参与mRNA输出的蛋白质利用输入途径返回细胞核，以便它们可以参与另一轮mRNA输出。我们使用裂殖酵母粟酒裂殖酵母（S. pombe）作为模型系统来研究真核细胞如何将其 mRNA 输出到细胞核外的机制。各种实验表明，酵母 Mex67/后生动物 Tap 和 Rae1 都参与 mRNA 成熟后步骤的 mRNA 输出，包括将 mRNA-蛋白质复合物连接到核孔的步骤。许多可溶性因子的身份，包括 Mex67 介导的 mRNA 输出途径的一些蛋白质-蛋白质相互作用，都是已知的。我们的研究旨在确定 Rae1p 介导的 mRNA 输出途径的蛋白质-蛋白质相互作用。我们发现，与酿酒酵母中的 Mex67/Mtr2 蛋白一起发挥作用的酿酒酵母 Yra1p、Sub2p、Sac3p 的同源物是粟酒裂殖酵母中 Rae1p 介导的 mRNA 输出途径的组成部分。在粟酒裂殖酵母中，它们分别被称为 Mlo3p、Uap56p 和 Sac3p。此外，Dss1p 是人乳腺癌肿瘤抑制因子 (BRAC2) 辅因子 DSS1 的粟酒裂殖酵母同源物，是一种与 Rae1p 一起发挥作用的新型 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 似乎充当 Mlo3p 和 Rae1p 之间的连接体。 Tap-tagged Dss1 可以在体内免疫沉淀相互作用的蛋白质，从而证实了体外数据。基于这些实验，我们开发了粟酒裂殖酵母中 Rae1p 介导的 mRNA 输出基本途径的机制模型。本质上，在这个模型中，Dss1p 和 Uap56p 可以通过桥接 Mlo3p 和 Rae1p 将 mRNA 连接到核孔。