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

植物RNA结合蛋白：功能基因组学的工具和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之间的相互作用奠定了植物发育和功能的每个方面。因此，植物基因组编码了700种含有预测的RNA结合基序的蛋白质，但这些蛋白质中很少有人研究过。为了剖析植物中的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，确定它们相互作用的RNA，并确定这些相互作用如何影响RNA和下游过程的命运。该项目开发了解决这些问题的工具，并将它们应用于一组40个RNA结合蛋白。由40个蛋白质组成的实验集强调了CRM和PPR蛋白质家族，这两个蛋白质家族主要是植物特有的，总共有大约500个成员，其中大多数被预测以叶绿体或线粒体为靶标。项目目标是：(I)开发一个预测的植物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。该数据库将以基因模型、细胞内靶向预测、实验数据和文献引用为注释的交叉引用的直系物和平行同源物为特色；(Ii)使用将免疫共沉淀与微阵列技术相结合的方法来识别测试集中的40种蛋白质中的每一种在体内与之相关的RNA；(Iii)通过分析由于玉米和水稻中基因表达减少而导致的分子缺陷，准确地确定受这40种蛋白质影响的RNA新陈代谢的方面；(Iv)测试一种提高插入Mu转座子引起的玉米突变克隆效率的方法，如果成功，将其应用于现有的一组Mu诱导的、叶绿体RNA代谢缺陷的突变体；(V)通过在两个地点的推广计划，提高社区对植物研究的认识。预期成果包括：(1)植物中RNA结合蛋白质的综合关系数据库，它将对广泛的植物生物学研究人员有价值；(2)通过确定特定的RNA底物和具有代表性的家族成员的生理作用，阐明最近发现的两个植物蛋白家族，即PPR和CRM家族的一般性质；(3)继续提供叶绿体生物发生基因的反向遗传筛选服务；(4)分别为大学生和高中生提供有关水稻转化和遗传学的教程，以及在Ithaca和Eugene实施K-12推广方案。通过该项目的光合作用突变体库和数据库可以访问工具和数据，网址为http://chloroplast.uoregon.edu/.

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/.