Molecular and Functional Dissection of Distinct mRNA Export Pathways

不同 mRNA 输出途径的分子和功能剖析

• 批准号: 10406919
• 负责人: Samson Obado
• 金额: $ 38.14万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-10 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10406919
• 关键词: Affinity; Biochemical; Biological; Biological Models; Biological Process; Blood Circulation; Carrier Proteins; Catalogs; Cell Nucleus; Cells; Cellular biology; Complex; Cytoplasm; Data; Disease; Dissection; Engineering; Environment; Eukaryota; Eukaryotic Cell; Evolution; Exercise; Exhibits; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Human; Hydrolysis; Individual; Insecta; Knowledge; Lead; Life; Life Cycle Stages; Maintenance; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Methods; Modeling; Molecular; Nuclear Pore Complex; Organ; Organism; Pathway interactions; Play; Post-Transcriptional Regulation; Process; Protein Isoforms; Proteins; Proteome; Proteomics; Protozoa; Publishing; RNA; RNA Interference; Role; Specificity; System; Techniques; Testing; Textbooks; Tissues; Transcript; Transcriptional Regulation; Trypanosoma; Untranslated Regions; Variant; Vertebrates; Yeasts; base; cell type; crosslink; driving force; genetic information; insight; mRNA Export; new therapeutic target; novel; novel therapeutic intervention; nucleocytoplasmic transport; paralogous gene; promoter; protein function; transcriptome sequencing; virtual

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.

项目概要 主要细胞 mRNA 输出因子 Mex67/NXF1（酵母/脊椎动物）从细胞核运输其货物 以独特的 ATP 依赖性途径通过核孔复合体 (NPC) 到达细胞质，即 与依赖于 GTP/GDP 的 Ran 循环的所有其他出口因素不同。在大多数真核生物中， Mex67/NXF1 作为单一蛋白质存在；然而，后生动物中存在 NXF1 的其他组织特异性亚型 （即人类）。由于这些组织特异性亚型的功能很难界定 后生动物基因表达控制的复杂性以及缺乏可能的简单模型系统 模仿不同的组织。我们最近发现锥体虫，一组不同的单细胞动物 原生动物有两个非常不同的 Mex67 旁系同源物，在 mRNA 输出中发挥不同的作用。锥虫科 缺乏个体基因启动子控制，而是严重依赖转录后基因调控 潜在地，将 RNA 输出置于基因表达控制的中心。确实，我们的初步结果 表明两个 Mex67 旁系同源物具有生命周期特异性作用，让人想起组织特异性 NXF1 在后生动物中观察到的变异。我们的假设是，这两个 Mex67 旁系同源物的功能有助于差异化地帮助 调控基因在不同生命周期阶段的表达。为了确定这一点，我们将定量和 全面表征两个 Mex67 的旁系同源蛋白相互作用组和 RNA 货物 通过利用和调整我们首创的方法，以及不同生命阶段细胞中的蛋白质 标准生化技术。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 缺乏完整的 ATP 依赖性输出机制 驱动酵母和后生动物中 mRNA 输出所需的。相反，我们的结果表明生物体依赖于 Ran GTPase 系统，与细胞内所有其他运输途径一样，与规范的重大背离 mRNA输出的教科书模型。我们假设该系统将为 Ran 如何进行提供新的视角 可用于介导跨 NPC 的定向传输。我们的策略将包括经典的生化 涉及外源表达成分的技术已成功用于界定 酵母和人类的核细胞质运输，以及最先进的蛋白质组学方法来计算 TbMex67-Ran 机器的拓扑图。我们的结果将有助于揭示不同的生物体和 组织可能利用输出作为控制基因表达的重要机制，以及Ran如何驱动 以以前未曾预料到的方式改变不同的核转运途径。