Messenger RNA transport across the nuclear pore complex

描述(申请人提供)：在所有真核生物中，大分子在细胞核和细胞质之间的运输是一个基本的细胞过程。真核生物基因组的维持和解码以及真核生物转录组和蛋白质组的动态依赖于核膜上大量蛋白质和RNA的区隔和交换。此外，核质转运的调控为信号转导途径和发育刺激调控真核生物差异基因表达提供了重要机制。此外，许多病毒以细胞核运输机制的组件为目标，并利用或修改它们来促进病毒传播。因此，更好地了解介导核质运输的分子机制对于了解基本的细胞过程和新的抗病毒疗法的开发都是至关重要的。尽管信使RNA(信使RNA)输出对于真核基因表达至关重要，但含有信使RNA的核糖核蛋白颗粒(RNPs)的包装、加工和运输的许多方面尚未被阐明。这项建议中描述的研究计划的长期目标是了解mRNAs被靶向核膜并通过核膜转移的分子途径。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的移位时释放其在细胞质中的货物。这种对货物结合和释放的空间调节对于mRNAs的运输很重要，但在机制水平上仍然知之甚少。此外，我们实验室和其他实验室获得的证据表明，可溶性的肌醇多聚磷酸盐在基因输出中发挥着重要作用，但这些效应物的靶标(S)尚未确定。因此，我们特别提出：(1)确定主要的Poly(A)结合蛋白Pab1在mRNA成熟和输出中的作用；(2)确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输出途径的中间产物，并确定mRNA输出复合体是如何在细胞质中分解的。拟议的实验利用了酿酒酵母中可用的蛋白质组和基因组学工具，并结合了创新的生化、遗传和细胞生物学方法来解决这三个特定目标。由于信使核糖核酸的运输是一个高度保守的过程，从这些研究中获得的机械性见解将直接与包括人类在内的所有真核生物相关。

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.