Messenger RNA transport across the nuclear pore complex

描述（由申请人提供）：大分子在细胞核和细胞质之间的转运是所有真核生物中必不可少的细胞过程。真核基因组的维持和解码以及真核转录组和蛋白质组的动态依赖于大量蛋白质和RNA跨核膜的区室化和交换。此外，它是有据可查的，核质转运的调节提供了一个重要的机制，信号转导途径和发育刺激控制真核生物中的差异基因表达。此外，许多病毒靶向细胞核转运机制的组分，并利用或修饰它们以促进病毒繁殖。因此，更好地了解介导核质转运的分子机制对于理解基本细胞过程和开发新的抗病毒疗法都是至关重要的。尽管信使RNA（mRNA）输出对于真核基因表达至关重要，但含有mRNA的核糖核蛋白颗粒（RNP）从细胞核的包装、加工和运输的许多方面尚未阐明。本提案中描述的研究计划的长期目标是了解mRNA靶向和跨核膜转运的分子途径。mRNA的输出似乎是由多种可溶性蛋白质因子介导的，所述可溶性蛋白质因子特异性结合细胞核中的mRNA，但在通过核孔复合物易位时在细胞质中释放其货物。这种货物结合和释放的空间调节对于mRNA的转运很重要，但在机制水平上仍然知之甚少。此外，在我们的实验室和其他实验室获得的证据表明，可溶性肌醇多磷酸在mRNA输出中起重要作用，但这些效应物的靶点尚未确定。因此，我们具体建议：（1）鉴定主要的poly（A）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、分离和鉴定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。拟议的实验利用了酵母酿酒酵母中可用的蛋白质组学和基因组学工具，并采用创新的生物化学，遗传学和细胞生物学方法相结合，以解决这三个具体目标。由于mRNA转运是一个高度保守的过程，从这些研究中获得的机制见解将直接与所有真核生物，包括人类。

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.