Molecular and Functional Dissection of Distinct mRNA Export Pathways

项目摘要 主要的细胞mRNA输出因子Mex 67/NXF 1（酵母/脊椎动物）从细胞核运输其货物 通过核孔复合体（NPC）以独特的ATP依赖性途径进入细胞质，即 与其他依赖GTP/GDP的Ran循环的出口因素不同。在大多数真核生物中， Mex 67/NXF 1以单一蛋白存在;然而，在后生动物中存在NXF 1的其他组织特异性同种型 (i.e.人类）。这些组织特异性同种型的功能一直难以界定，这是由于 后生动物基因表达控制的复杂性以及缺乏简单的模型系统， 模仿不同的组织我们最近发现，锥虫，一组分歧的单细胞 原生动物，有两个非常不同的旁系同源的Mex 67具有不同的作用，在mRNA输出。锥体虫 缺乏单个基因启动子控制，而是严重依赖于转录后基因调控， 潜在地，将RNA输出置于基因表达控制的中心。事实上，我们的初步结果 表明两个Mex 67旁系同源物具有生命周期特异性作用，使人想起组织特异性NXF 1 在后生动物中观察到的变体。我们的假设是，这两个Mex 67旁系同源物的功能，以帮助差异 在不同的生命周期阶段调节基因的表达。为了确定这一点，我们将定量和 全面表征两个Mex 67的旁系同源特异性蛋白质相互作用组和RNA货物 蛋白质在不同的生命阶段的细胞通过利用和适应的方法，我们已经开创，以及 标准生化技术。随着核转运在基因调控中作用的扩大， 我们以前已经证明，锥虫NPC缺乏整个依赖ATP的输出机制 在酵母和后生动物中驱动mRNA输出。相反，我们的研究结果表明，生物体依赖于 然GT3系统，像所有其他运输途径内的细胞，一个重大的偏离规范 mRNA输出的教科书模型。我们假设，这个系统将提供一个新的视角，如何运行 可用于介导穿过NPC的定向运输。我们的策略将包括经典的生化 涉及外源表达组分的技术已成功用于界定 酵母和人类的核质转运，以及最先进的蛋白质组学方法来计算 TbMex 67-Ran机器的拓扑图。我们的研究结果将有助于揭示不同的生物和 组织可能使用输出作为控制基因表达的重要机制，以及Ran如何驱动 不同的核运输途径以前所未有的方式。

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.