Arabidopsis 2010: Global Analysis of Translational Regulons

这个项目将为任何真核生物做出第一个全面的努力，以定义其表达受一般翻译启动机制调控的基因。有证据表明，翻译起始的基因特异性调控是广泛存在的，在系统发育上是保守的，并在各种环境和发育条件下发生。真核细胞起始因子(EIF)复合体个别亚基的突变破坏了基因特异的翻译控制通路，但关于翻译调控的程度及其对特定EIF的依赖还没有全面的信息。在我们的初步数据和互补专业知识的基础上，该项目将定义其mRNAs共同依赖于特定翻译因素的基因组，即“翻译调节因子”。这项工作将通过追求三个具体目标提供第一个全基因组范围的翻译调控通路网络的概述：1)确定eIF突变体的发育、生理和应激相关的表型。将在大体形态和发育、对非生物胁迫(例如，热)、光和光合作用的反应水平上评估受特定翻译因子丢失影响的代谢和发育途径。2)确定其翻译受特定EIF控制的候选客户mRNAs。对来自eIF突变体的多体RNA进行DNA微阵列分析，以确定每个eIF的客户mRNAs。对客户mRNAs的详细生物信息学分析将被用来定义翻译调节因子，并预测可能影响翻译调节的mRNA结构和/或序列特征。3)EIF和客户mRNA翻译调节的有效性。在翻译客户端mRNA子集时对EIF的要求将使用体内mRNA报告程序构建来验证。拟南芥的体外翻译裂解物将用于在同源的体外翻译分析中分析客户mRNAs对eIF的依赖。从更广泛的影响来看，这个项目将极大地增加我们对植物翻译控制的理解，并作为真核生物的第一个全基因组努力，将对植物科学和更广泛的科学界做出相当大的贡献。利用加州大学河滨分校、德克萨斯大学奥斯汀分校、德克萨斯大学诺克斯维尔分校和亚利桑那大学拥有大量少数族裔学生这一事实，该项目将包括一个强有力的教育部分，强调少数族裔研究生和本科生参与科学研究，鼓励少数族裔学生从事科学研究、教育或政策事业。研究生和博士后将对该项目进行全日制研究。本科生将通过NSF支持的新生研究倡议(UT-Austin)或本科生生物学研究计划(U-Arizona)参与跨学科培训，前者为新生提供重要的研究经验，后者将本科生安排到研究实验室。该项目还将与联邦政府资助的促进少数族裔科学培训的项目合作，包括NSF资助的加州少数族裔参与科学、工程和数学联盟(CAMP-UCR)，该联盟致力于将获得科学学位的少数族裔学生的数量增加一倍；哥白尼项目，这是美国教育部的一个计划，旨在大幅增加科学教师的数量、质量和多样性(UC-Riverside)；以及NSF资助的路易丝·斯托克斯少数族裔参与联盟，该联盟将来自田纳西州立大学其他系统学校的少数族裔学生吸引到夏季进行研究。此外，每个实验室将使用夏季REU补充品，为更多的本科生提供研究经验。拟议的研究包括适合各级学生(高中、本科生、实习生、研究生和博士后)参与的研究方法，从而提供积极的研究经验，同时培养具有科学素养的公民，并产生关于翻译机制在调节基因表达方面的作用的广泛信息。所有项目数据和生物资源都可以通过菲亚特项目网站(http://research.cm.utexas.edu/kbrowning/fiat/).获取突变和相关的表型数据和图像也将通过拟南芥生物资源中心获得，而微阵列数据将保存并通过NCBI基因表达总览(GEO，http://www.ncbi.nlm.nih.gov/geo/).)获得

This project will make the first comprehensive effort, for any eukaryote, toward defining genes whose expression is regulated by the general translation initiation machinery. Evidence indicates that gene-specific regulation of translation initiation is widespread, phylogenetically conserved, and occurs under a variety of environmental and developmental conditions. Gene-specific pathways of translational control are disrupted by mutations in individual subunits of the eukaryotic initiation factor (eIF) complexes, but there is no comprehensive information about the extent of translational regulation and its dependence on specific eIFs. Building on our preliminary data and complementary expertise, this project will define groups of genes whose mRNAs are co-dependent on specific translation factors, i.e. "translational regulons". This work will provide the first genome-wide overview of the network of translational regulatory pathways through the pursuit of three specific aims:1) Determine the developmental, physiological, and stress-related phenotypes of eif mutants. The metabolic and developmental pathways impacted by the loss of a specific translation factor will be assessed at the level of gross morphology and development, responses to an abiotic stress (i.e., heat), light, and photosynthesis.2) Identify candidate client mRNAs whose translation is controlled by specific eIFs. DNA microarray analysis on polysomal RNA from eif mutants will be performed to identify client mRNAs for each eIF. Detailed bioinformatic analyses of the client mRNAs will be used to define translational regulons and to predict mRNA structural and/or sequence features that may influence translational regulation.3) Validation of eIF and client mRNA translational regulation. The requirement of an eIF in the translation of a subset of client mRNAs will be validated using in vivo mRNA-reporter constructs. An in vitro translation lysate will be developed from Arabidopsis to be used in the analysis of the eIF-dependence of client mRNAs in a homologous in vitro translation assay.With respect to broader impacts, this project will dramatically increase our understanding of translational control in plants and as the first genome-wide effort for any eukaryote and will make a considerable contribution to plant science and to the greater scientific community. Taking advantage of the fact that UC-Riverside, UT-Austin, UT-Knoxville, and U-Arizona have substantial representation of minority students, the project will involve a robust educational component emphasizing the involvement of minority graduate and undergraduate students in scientific research, acting to encourage minority students to pursue a career in scientific research, education, or policy. Graduate and post-doctoral students will be engaged in full-time research on the project. Undergraduate students will be involved in cross-disciplinary training through the NSF-supported Freshman Research Initiative (UT-Austin), which provides freshmen a significant research experience, or the Undergraduate Biology Research Program (U-Arizona) that places undergraduate students into research laboratories. The project will also partner with federally-funded programs that promote science training for minorities including the NSF-funded California Alliance for Minority Participation in Science, Engineering and Mathematics (CAMP-UCR) that works to double the number of minority students receiving a degree in science; the Copernicus Project, a U.S. Department of Education program that seeks to increase substantially the number, quality and diversity of science teachers (UC-Riverside); and the NSF-funded Louise Stokes Alliance for Minority Participation, which brings minority students from other U-Tennessee system schools to do summer research. In addition, each laboratory will use summer REU supplements to provide research experience to additional undergraduate students. The proposed research includes research approaches that are suitable for the involvement of students at every level (high school, undergraduate, intern, graduate and post-doctoral), thus providing a positive research experience while producing scientifically literate citizens as well as generating extensive information about the role of translational machinery in regulating gene expression. All project data and biological resources can be accessed through FIAT, the project website (http://research.cm.utexas.edu/kbrowning/fiat/). Mutants and associated phenotypic data and images will also be available through the Arabidopsis Biological Resource Center while microarray data will be deposited and available through the NCBI Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/).