Plant RNA Binding Proteins: Tools for Functional Genomics and Application to Chloroplast Biogenesis

植物RNA结合蛋白：功能基因组学工具及其在叶绿体生物发生中的应用PI：Alice Barkan，俄勒冈大学PI：Maureen Hanson，康奈尔大学蛋白质和RNA之间的相互作用是植物发育和功能的各个方面的基础。因此，植物基因组编码700种蛋白质，这些蛋白质具有预测的RNA结合基序，但很少有人研究这些蛋白质。为了剖析植物中RNA-蛋白质相互作用网络，有必要对植物RNA结合蛋白进行分类，以识别与它们相互作用的RNA，并确定这些相互作用如何影响RNA的命运和下游过程。该项目开发了解决这些问题的工具，并将其应用于一组40种RNA结合蛋白。40种蛋白质的实验组强调CRM和PPR蛋白质家族，它们主要是植物特有的，总共包含约500个成员，其中大部分被预测为靶向叶绿体或线粒体。项目目标是：（i）开发一个预测植物RNA结合蛋白的关系数据库，该数据库将整合来自水稻、玉米和拟南芥的数据。该数据库将包括交叉引用的直系同源物和旁系同源物，并附有基因模型、细胞内靶向预测、实验数据和文献引用; (ii)使用一种将免疫共沉淀与微阵列技术相结合的方法，以鉴定测试集中的40种蛋白质中的每一种蛋白质在体内与之相关的RNA; ㈢通过分析玉米和水稻中因其基因表达减少而导致的分子缺陷，查明受这40种蛋白质影响的RNA代谢的各个方面; ㈣测试一种方法，以提高克隆Mu转座子插入引起的玉米突变的效率，如果成功，将其应用于现有的一组叶绿体RNA代谢缺陷的Mu诱导突变体;（v）通过在两个地点开展外联方案，提高社区对植物研究的认识。 预期成果包括：（i）一个全面的植物RNA结合蛋白关系数据库，这将对广泛的植物生物学研究人员具有价值;（ii）通过鉴定特定的RNA底物和代表性家族成员的生理作用，阐明两个最近被认可的植物蛋白家族PPR和CRM家族的一般性质;（iii）继续对叶绿体生物发生基因进行反向遗传筛选服务;（iv）分别为大学生和高中生提供水稻转化和遗传学教程，以及伊萨卡和尤金的K-12外展计划。可通过该项目的光合突变体图书馆和数据库http://chloroplast.uoregon.edu/获得工具和数据。

Plant RNA Binding Proteins: Tools for functional genomics and application to chloroplast biogenesisPI: Alice Barkan, University of OregoncoPI: Maureen Hanson, Cornell UniversityInteractions between proteins and RNAs underlie every aspect of plant development and function. Accordingly, plant genomes encode 700 proteins that harbor predicted RNA binding motifs, but few of these proteins have been studied. To dissect RNA-protein interaction networks in plants, it is necessary to catalog plant RNA-binding proteins, to identify the RNAs with which they interact, and to determine how those interactions influence the fate of the RNA and downstream processes. This project develops tools to address each of these issues, and applies them to a set of 40 RNA binding proteins. The experimental set of 40 proteins emphasizes the CRM and PPR protein families, which are largely specific to plants and which together contain ~500 members, most of which are predicted to be targeted to chloroplasts or mitochondria. Project goals are to: (i) Develop a relational database of predicted plant RNA binding proteins that will integrate data from rice, maize, and Arabidopsis. The database will feature cross-referenced orthologs and paralogs, annotated with gene models, intracellular targeting predictions, experimental data and literature citations; (ii) Use an approach that couples coimmunoprecipitation with microarray technology to identify RNAs with which each of the 40 proteins in the test set is associated in vivo; (iii) Pinpoint aspects of RNA metabolism that are influenced by these 40 proteins, through the analysis of molecular defects resulting from reduced expression of their genes in maize and rice; (iv) Test an approach to increase the efficiency of cloning maize mutations caused by insertions of Mu transposons and, if successful, apply it to an existing set of Mu-induced mutants with defects in chloroplast RNA metabolism; (v) Increase community awareness of plant research through outreach programs at both sites. Expected outcomes include (i) a comprehensive relational database of RNA-binding proteins in plants, which will be of value for a broad range of plant biology researchers; (ii) elucidation of the general properties of two recently recognized plant protein families, the PPR and CRM families, through the identification of specific RNA substrates and physiological roles for representative family members; (iii) continuation of a reverse-genetic screening service for chloroplast biogenesis genes; (iv) tutorials on rice transformation and genetics for college and high school students, respectively, and K-12 outreach programs in Ithaca and Eugene. Access to tools and data is available through the project's Photosynthetic Mutant Library and database at http://chloroplast.uoregon.edu/.