Arabidopsis 2010: Global Analysis of Translational Regulons

该项目将为任何真核生物做出第一次全面的努力，以确定其表达受一般翻译起始机制调控的基因。有证据表明，翻译起始的基因特异性调控是广泛的，非遗传保守的，并发生在各种环境和发育条件下。基因特异性的翻译控制途径被真核起始因子复合物中单个亚基的突变所破坏，但关于翻译调控的程度及其对特定eIF的依赖性还没有全面的信息。基于我们的初步数据和互补的专业知识，该项目将定义其mRNA共同依赖于特定翻译因子（即“翻译调节子”）的基因组。这项工作将提供第一个全基因组的翻译调控途径网络的概述，通过追求三个具体的目标：1）确定发育，生理和压力相关的表型eif突变体。将在总体形态和发育水平、对非生物胁迫的反应（即，2）鉴定其翻译由特定eIF控制的候选客户mRNA。将对eif突变体的多聚体RNA进行DNA微阵列分析，以鉴定每个eIF的客户mRNA。对客户mRNA进行详细的生物信息学分析将用于定义翻译调节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翻译调节的验证。将使用体内mRNA报告基因构建体验证在客户mRNA子集的翻译中eIF的需求。将从拟南芥中开发一种体外翻译裂解物，用于在同源体外翻译测定中分析客户mRNA的eIF依赖性。就更广泛的影响而言，该项目将极大地提高我们对植物翻译控制的理解，并作为任何真核生物的第一个全基因组努力，将为植物科学和更大的科学界做出相当大的贡献。利用加州大学河滨分校，德州大学奥斯汀分校，德州大学诺克斯维尔分校和亚利桑那大学有大量少数民族学生的代表性，该项目将涉及一个强大的教育组成部分，强调少数民族研究生和本科生参与科学研究，鼓励少数民族学生从事科学研究，教育或政策。研究生和博士后学生将全职从事该项目的研究。本科生将通过NSF支持的新生研究计划（UT-Austin）参与跨学科培训，该计划为新生提供重要的研究经验，或将本科生置于研究实验室的本科生生物学研究计划（U-Arizona）。该项目还将与联邦资助的促进少数民族科学培训的项目合作，包括NSF资助的加州少数民族参与科学、工程和数学联盟（CAMP-UCR）致力于使获得科学学位的少数民族学生人数增加一倍;哥白尼计划是美国教育部的一个项目，旨在大幅增加这一数字，科学教师的质量和多样性（加州大学河滨分校）;和NSF资助的路易丝斯托克斯少数民族参与联盟，它带来了其他U-田纳西系统学校的少数民族学生做暑期研究。此外，每个实验室将使用夏季REU补充提供研究经验，以额外的本科生。拟议的研究包括适合各级学生（高中，本科，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，同时产生科学素养的公民，以及生成有关翻译机制在调节基因表达中的作用的广泛信息。所有项目数据和生物资源均可通过FIAT项目网站（http：//research.cm.utexas.edu/kbrowning/fiat/）查阅。突变体和相关的表型数据和图像也可通过拟南芥生物资源中心获得，而微阵列数据将通过NCBI基因表达综合数据库（GEO，http：//www.ncbi.nlm.nih.gov/geo/）保存和获得。

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/).