Mechanism and regulation of mRNA export

mRNA输出的机制和调控

• 批准号: 6950119
• 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/6950119
• 关键词: HeLa cells; Schizosaccharomyces pombe; biological models; cell growth regulation; fungal genetics; gene expression; immunoprecipitation; intracellular transport; messenger RNA; nuclear membrane; protein protein interaction; protein transport; ribonucleoproteins; transport proteins

In eukaryotic cells, the process of the nuclear export of messenger RNA (mRNA) physically and functionally couple nuclear gene transcription and mRNA splicing with the translation apparatus in the cytoplasm. Fully mature transcripts are exported as ribonucleoprotein (RNP) complexes through specialized pores embedded within the nuclear envelope. It is thought that one or more proteins within the RNP act as carriers to target the mRNA to the pores and mediate its exit from the nucleus by interacting with various pore-associated proteins. In the cytoplasm, the RNP complex is disassembled to release the mRNA for translation; the proteins involved in export are returned to the nucleus for initiating another round of mRNA export. We use the fission yeast Schizosaccharomyces pombe (S. pombe) and employ genetic and biochemical approaches to understand the functions of two key conserved mRNA export factors, Rae1p (Gle2 in metazoan or S. cerevisiae) and the NXF/NXT (Mex67/p15 in S. pombe or Tap/p15 in metazoan) heterodimer. In S. pombe, the role of Rae1p in mRNA export is essential. The functions of NXF/NXT homolog in various organisms appear to be essential in mRNA export, but surprisingly in S. pombe, the role of their homolog Mex67p/p15 is redundant. Both Rae1p and Mex67p/p15 are thought to function at the terminal nuclear steps of mRNA export presumably for linking RNP complexes to the pore and for mediating RNP export through the nuclear pore complexes (NPC). In the Mex67p/Tap mediated pathway, an early mRNA export factor Sub2p (metazoan UAP56) is recruited to activated genes and the transcripts and Sub2p in turn recruits RNA annealing protein Yra1p (Mlo3 in S. pombe and Aly in metazoan) to the nascent messages. Mex67/Tap is believed to displace Sub2p (UAP56) from the RNP complex to bind Yra1p (Aly). Interactions between Mex67p/Tap with ?FG (Phelylalanine-Glycine repeat) containing nucleoporins are believed to underlie pore targeting as well as the passage of the RNP cargo through the NPC. No similar mechanistic details are known about how Rae1p functions. We have identified esl49 as a gene whose functions are crucial for mediating mRNA export via Rae1p. The encoded protein is conserved across evolution. We have found that Els49p interacts directly with Mlo3p (Yra1p) and with Rae1p while Mlo3p does not interact with Rae1p directly. A ternary complex containing Mlo3p-Esl49p-Rae1p readily forms in vitro demonstrating Esl49p could link Mlo3p to Rae1p. Immuno-precipitation experiments suggest that Esl49p is associated with Mlo3p and with Rae1p in complexes in vivo. These studies have uncovered the mRNA export components functioning in the Rae1p-mediated essential mRNA export pathway of S. pombe. To understand how NXF proteins could be targeted to the nuclear pore, we have identified additional pore targeting domains in analogous regions in spMex67p and hTap by genetic protein export assays in S. pombe and in the HeLa cells. These domains are not part of the two FG interacting pockets known in Mex67p/hTap. We have further found direct in vitro binding of ?FG containing regions of Nup98 and Nup159 with these pore-targeting domains. Mutations that abolished nuclear export properties of these domains in vivo concomitantly led to loss of interaction with the ?FG proteins. These results lead us to propose the existence of new and conserved pore targeting domains in human and S. pombe NXF homologs.

在真核细胞中，信使 RNA (mRNA) 的核输出过程在物理上和功能上将核基因转录和 mRNA 剪接与细胞质中的翻译装置耦合。完全成熟的转录物通过嵌入核膜内的特殊孔作为核糖核蛋白（RNP）复合物输出。据认为，RNP 内的一种或多种蛋白质充当载体，将 mRNA 靶向孔，并通过与各种孔相关蛋白质相互作用介导其从细胞核中退出。在细胞质中，RNP复合物被分解，释放出mRNA进行翻译；参与输出的蛋白质返回细胞核以启动另一轮 mRNA 输出。 我们使用裂殖酵母粟酒裂殖酵母 (S. pombe) 并采用遗传和生化方法来了解两个关键保守 mRNA 输出因子 Rae1p（后生动物或酿酒酵母中的 Gle2）和 NXF/NXT（酿酒酵母中的 Mex67/p15）的功能。粟酒裂殖酵母或后生动物中的 Tap/p15) 异二聚体。在粟酒裂殖酵母中，Rae1p 在 mRNA 输出中的作用至关重要。 NXF/NXT 同源物在各种生物体中的功能似乎对于 mRNA 输出至关重要，但令人惊讶的是，在粟酒裂殖酵母中，其同源物 Mex67p/p15 的作用是多余的。 Rae1p 和 Mex67p/p15 均被认为在 mRNA 输出的末端核步骤中起作用，可能用于将 RNP 复合物连接到孔并介导通过核孔复合物 (NPC) 的 RNP 输出。在 Mex67p/Tap 介导的途径中，早期 mRNA 输出因子 Sub2p（后生动物 UAP56）被招募到激活的基因和转录本中，而 Sub2p 反过来又将 RNA 退火蛋白 Yra1p（粟酒裂殖酵母中的 Mlo3 和后生动物中的 Aly）招募到新生信息中。 。 Mex67/Tap 被认为可从 RNP 复合物中取代 Sub2p (UAP56) 以结合 Yra1p (Aly)。 Mex67p/Tap 与含有核孔蛋白的 ?FG（苯丙氨酸-甘氨酸重复序列）之间的相互作用被认为是孔靶​​向以及 RNP 货物通过 NPC 的基础。关于 Rae1p 的功能尚无类似的机制细节。我们已经确定 esl49 是一个基因，其功能对于通过 Rae1p 介导 mRNA 输出至关重要。编码的蛋白质在进化过程中是保守的。我们发现 Els49p 直接与 Mlo3p (Yra1p) 和 Rae1p 相互作用，而 Mlo3p 不直接与 Rae1p 相互作用。含有 Mlo3p-Esl49p-Rae1p 的三元复合物在体外很容易形成，证明 Esl49p 可以将 Mlo3p 与 Rae1p 连接起来。免疫沉淀实验表明，Esl49p 在体内复合物中与 Mlo3p 和 Rae1p 相关。这些研究揭示了在 Rae1p 介导的粟酒裂殖酵母必需 mRNA 输出途径中起作用的 mRNA 输出组件。 为了了解 NXF 蛋白如何靶向核孔，我们通过在粟酒裂殖酵母和 HeLa 细胞中进行遗传蛋白输出测定，在 spMex67p 和 hTap 的类似区域中鉴定了额外的孔靶向结构域。这些结构域不是 Mex67p/hTap 中已知的两个 FG 相互作用口袋的一部分。我们进一步发现含有 Nup98 和 Nup159 区域的 ?FG 与这些孔靶向结构域在体外直接结合。体内废除这些结构域的核输出特性的突变同时导致与?FG蛋白相互作用的丧失。这些结果使我们提出人类和粟酒裂殖酵母 NXF 同源物中存在新的保守孔靶向结构域。