Molecular and Functional Dissection of Distinct mRNA Export Pathways

项目摘要 主要的细胞mRNA输出因子Mex67/Nxf1(酵母/脊椎动物)从细胞核运输其货物 通过核孔复合体(NPC)进入细胞质，这是一条明显的ATP依赖途径，即 与依赖GTP/GDP的RAN周期依赖的所有其他出口因素不同。在大多数真核生物中， Mex67/Nxf1以单一蛋白质形式存在；然而，Nxf1的其他组织特异性亚型存在于后生动物中 (即人类)。这些组织特异性异构体的功能很难界定，因为 后生动物基因表达控制的复杂性，以及缺乏可能 模仿不同的组织。我们最近发现，锥虫，一组发散的单细胞 原生动物，有两个非常不同的Mex67对偶基因，在信使核糖核酸输出中扮演不同的角色。锥虫 缺乏对单个基因启动子的控制，而是严重依赖转录后基因调控和 潜在地，将RNA输出置于基因表达控制的中心。的确，我们的初步结果 表明两个Mex67类似物具有特定于生命周期的作用，这让人想起组织特定的Nxf1 在后生动物中观察到的变种。我们的假设是，这两个Mex67对偶基因的作用是不同的 调控不同生命周期阶段的基因表达。为了确定这一点，我们将从数量和 两株Mex67的Paralog特异性蛋白相互作用和RNA产物的综合特征 在不同的生命阶段，通过利用和适应我们开创的方法，以及 标准的生化技术。伴随着核运输在基因调控中作用的扩大， 我们之前已经证明，锥虫NPC缺乏依赖于ATP的整个出口机制 在酵母和后生动物中驱动信使核糖核酸的输出。相反，我们的结果表明，生物体依赖于 Ran GTP酶系统，像细胞内所有其他运输途径一样，重大背离了规范 信使核糖核酸输出的教科书模型。我们假设该系统将提供有关如何运行的新视角 可以用来调解通过NPC的定向运输。我们的战略将包括经典生化 涉及外源表达成分的技术，这些成分已被成功地用于划分 酵母和人类的核质运输，以及最新的蛋白质组学方法 TbMex67-RAN机械的拓扑图。我们的结果将有助于揭示不同的生物体和 组织可能将出口作为控制基因表达的重要机制，以及RAN可能如何驱动 不同的核运输途径以前没有预料到的方式。

Project Summary The major cellular mRNA export factor Mex67/NXF1 (yeast/vertebrates) transports its cargo from the nucleus through the nuclear pore complex (NPC) to the cytoplasm in a distinct ATP dependent pathway, that is different from all other export factors that rely on the GTP/GDP-dependent Ran cycle. In most eukaryotes, Mex67/NXF1 exists as a single protein; however, additional tissue-specific isoforms of NXF1 exist in metazoa (i.e. humans). The functions of these tissue-specific isoforms have been difficult to delimit due to the complexity of control of gene expression in metazoa as well as the lack of simple model systems that may mimic different tissues. We recently discovered that Trypanosomatids, a group of divergent unicellular protozoans, have two very distinct paralogs of Mex67 with differing roles in mRNA export. Trypanosomatids lack individual gene promoter control, instead relying heavily on post-transcriptional gene regulation and potentially, placing RNA export at the center of the control of gene expression. Indeed, our preliminary results indicate that the two Mex67 paralogs have life cycle-specific roles, reminiscent of the tissue-specific NXF1 variants observed in metazoa. It is our hypothesis that these two Mex67 paralogs function to help differentially regulate the expression of genes in different life cycle stages. To determine this, we will quantitatively and comprehensively characterize the paralog-specific protein interactomes and RNA cargos of the two Mex67 proteins in cells in the different life stages by utilizing and adapting methods we have pioneered, as well as standard biochemical techniques. Concomitant with an expanded role of nuclear transport in gene regulation, we have previously shown that the trypanosomatid NPC lacks the entire ATP-dependent export machinery required to drive mRNA export in yeast and metazoa. Instead, our results suggest organisms depend on the Ran GTPase system, like all other transport pathways within the cell, a major departure from the canonical textbook model of mRNA export. We hypothesize that this system will provide a new perspective on how Ran can be utilized to mediate directional transport across the NPC. Our strategy will include classical biochemical techniques involving exogenously expressed components that have been succesfully employed to delimit nucleocytoplasmic transport in yeast and humans, as well as state of the art proteomic methods to compute topological maps of the TbMex67-Ran machinery. Our results will help reveal how different organisms and tissues may use export as an important mechanism to control gene expression, and how Ran may drive different nuclear transport pathways in previously unanticipated ways.