Messenger RNA transport across the nuclear pore complex

信使 RNA 穿过核孔复合体的运输

• 批准号: 7118803
• 负责人: KARSTEN WEIS
• 金额: $ 27.16万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118803
• 关键词: RNA binding protein; Saccharomyces cerevisiae; biological signal transduction; fungal genetics; guanosinetriphosphatases; helicase; in situ hybridization; intracellular transport; messenger RNA; molecular cloning; nuclear membrane; nucleic acid sequence; protein structure function; protein transport; site directed mutagenesis; transport proteins

DESCRIPTION (provided by applicant): Transport of macromolecules between the nucleus and the cytoplasm is an essential cellular process in all eukaryotes. The maintenance and decoding of the eukaryotic genome, and the dynamic state of the eukaryotic transcriptome and proteome relies on the compartmentalization and exchange of a large number of proteins and RNAs across the nuclear envelope. Furthermore, it is well documented that the regulation of nucleocytoplasmic transport provides an important mechanism by which signal transduction pathways and developmental stimuli control differential gene expression in eukaryotes. In addition, many viruses target components of the cellular nuclear transport machinery, and exploit or modify them to promote viral propagation. Therefore, a better understanding of the molecular machinery that mediates nucleocytoplasmic transport is essential both for understanding fundamental cellular processes and the development of novel anti-viral therapies. Despite the critical importance of messenger RNA (mRNA) export for eukaryotic gene expression, many aspects of the packaging, processing, and transport of mRNA-containing ribonucleoprotein particles (RNPs) from the nucleus have not yet been elucidated. The long-term objective of the research program described in this proposal is to understand the molecular pathway by which mRNAs are targeted to and translocated across the nuclear envelope. Export of mRNA appears to be mediated by multiple soluble protein factors that specifically bind to mRNA in the nucleus but release their cargo in the cytoplasm upon translocation through the nuclear pore complex. This spatial regulation of cargo binding and release is important for the transport of mRNAs but remains poorly understood at the mechanistic level. Moreover, evidence obtained in our laboratory and others indicates an important role for soluble, inositol polyphosphates in mRNA export, but the target(s) of these effectors have not been identified. Thus, we specifically propose: (1) to characterize the role of the major poly (A)-binding protein Pabl in mRNA maturation and export; (2) to identify the function and the targets of soluble inositol polyphosphates in mRNA export; and, (3) to trap, isolate, and characterize intermediates of the mRNA export pathway and to determine how mRNA export complexes are disassembled in the cytoplasm. The proposed experiments take advantage of the proteomic and genomic tools available in the yeast Saccharomyces cerevisiae and employ a combination of innovative biochemical, genetic and cell biological approaches to address these three specific aims. Because mRNA transport is a highly conserved process, the mechanistic insights obtained from these studies will be directly relevant to all eukaryotes, including humans.

描述（由申请人提供）：细胞核和细胞质之间大分子的运输是所有真核生物中重要的细胞过程。真核基因组的维护和解码，以及真核转录组和蛋白质组的动态依赖于核膜上大量蛋白质和RNA的划分和交换。此外，有充分证据表明，核质运输的调节提供了信号转导途径和发育刺激控制真核生物差异基因表达的重要机制。此外，许多病毒以细胞核运输机制的成分为目标，并利用或修改它们来促进病毒繁殖。因此，更好地了解介导核细胞质运输的分子机制对于理解基本细胞过程和开发新型抗病毒疗法至关重要。尽管信使 RNA (mRNA) 输出对于真核基因表达至关重要，但从细胞核包装、加工和运输含有 mRNA 的核糖核蛋白颗粒 (RNP) 的许多方面尚未阐明。该提案中描述的研究计划的长期目标是了解 mRNA 靶向并跨核膜易位的分子途径。 mRNA 的输出似乎是由多种可溶性蛋白因子介导的，这些蛋白因子特异性地与细胞核中的 mRNA 结合，但在通过核孔复合体易位时将其货物释放到细胞质中。这种货物结合和释放的空间调节对于 mRNA 的运输很重要，但在机制层面仍然知之甚少。此外，我们实验室和其他实验室获得的证据表明可溶性肌醇多磷酸盐在 mRNA 输出中发挥着重要作用，但这些效应器的靶点尚未确定。因此，我们特别建议：（1）表征主要的多聚腺苷酸结合蛋白Pabl在mRNA成熟和输出中的作用； (2) 鉴定可溶性肌醇多磷酸在mRNA输出中的功能和靶点； (3) 捕获、分离和表征 mRNA 输出途径的中间体，并确定 mRNA 输出复合物如何在细胞质中分解。拟议的实验利用酿酒酵母中可用的蛋白质组和基因组工具，并采用创新的生化、遗传和细胞生物学方法的组合来解决这三个具体目标。由于 mRNA 运输是一个高度保守的过程，因此从这些研究中获得的机制见解将与包括人类在内的所有真核生物直接相关。