Collaborative Research: Role of Arabidopsis CTD-Phosphatase-Like 1 in Gene Silencing Pathways

合作研究：拟南芥 CTD-磷酸酶样 1 在基因沉默途径中的作用

• 批准号: 0950242
• 负责人: Jianhua Zhu
• 金额: $ 38.75万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-15 至 2016-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0950242&HistoricalAwards=false
• 关键词: Collaborative; Research; Role; Arabidopsis; CTD

Intellectual meritPlant gene expression is a dynamic process that involves positive and negative regulation at multiple levels. Recent studies emphasize importance of RNA/gene-silencing mechanisms mediated by small RNA molecules, which can trigger both transcriptional and posttranscriptional gene silencing not only in plants but also in other eukaryotes. Central to the small RNA production are proteins containing double-stranded (ds) RNA binding motif (DRM). Among eukaryotes, plants contain unique DRM-containing proteins. Carboxyl-terminal domain (CTD) phosphatase-like 1 (CPL1) proteins contain an N-terminal phosphatase domain and tandemly repeated C-terminal DRM domains. The primary hypothesis to be tested in this project is that a plant-specific CPL protein fine-tunes the level of gene silencing mediated by small RNA. Since CTD phosphorylation can regulate the activity of the pol II complex, our results imply that plants evolved an additional fine-tuning mechanism of gene expression, which uses components of transcription elongation for gene silencing. This project will dissect the interaction between transcription elongation machineries and gene silencing by determining roles of CPL1 in gene silencing. The specific aims of this project are: 1) to establish CPL1 structure essential for in vivo function: essential domains of CPL1 protein will be determined by genetic complementation assays using modified CPL1 genes, 2) to establish interaction between CPL1 and HYL1; the mode of CPL1-HYL1 interaction will be determined and its relationship with CPL1 function will be analyzed, 3) to determine the RNA/gene silencing mechanism of CPL1; effect of cpl1 mutation in DNA methylation, histone modifications and small RNA accumulation/action will be analyzed using molecular genetic analysis as well as transient RNAi analysis. The body of information to be generated in this project therefore will reveal novel mechanism of plant gene regulation that will also have implications in other organisms.Broader impactsThis project will be conducted by a two-institute team consisting of two post-doctoral associates and a graduate student with support from undergraduate student(s). The participants will be trained for understanding and effective use of plant genome information and various genetic resources as well as molecular biochemical skills to validate the information. To train future scientists from diverse backgrounds, a graduate-level laboratory module for the course Physiol. Mol. Basis of Plant Stress Responses, in which students will have hands-on experience, will be taught in TAMU, some component of which will be shared in the similar course in UM. This course has been taken by numerous students in the field of field-oriented agriculture, therefore, should provide basic knowledge for sustainable agricultural practices. Undergraduate students will participate in the research program using REU supplements. An international exchange program has been initiated and is being developed with Gyeongsang Natl Univ, S. Korea, where visiting scholars and students can participate in mutual research projects. This is an excellent opportunity for students to be exposed to multi-disciplinary and multiculture research environment.

智力优点植物基因表达是一个动态过程，涉及多个水平的正向和负向调节。 最近的研究强调了小RNA分子介导的RNA/基因沉默机制的重要性，它不仅可以在植物中而且在其他真核生物中触发转录和转录后基因沉默。 小 RNA 生产的核心是含有双链 (ds) RNA 结合基序 (DRM) 的蛋白质。 在真核生物中，植物含有独特的含 DRM 的蛋白质。 羧基末端结构域 (CTD) 磷酸酶样 1 (CPL1) 蛋白包含 N 末端磷酸酶结构域和串联重复的 C 末端 DRM 结构域。该项目要测试的主要假设是植物特异性 CPL 蛋白可微调小 RNA 介导的基因沉默水平。 由于 CTD 磷酸化可以调节 pol II 复合物的活性，因此我们的结果意味着植物进化出了一种额外的基因表达微调机制，该机制利用转录延伸的成分来实现基因沉默。 该项目将通过确定 CPL1 在基因沉默中的作用来剖析转录延伸机制与基因沉默之间的相互作用。 该项目的具体目标是：1）建立体内功能所必需的CPL1结构：CPL1蛋白的基本结构域将通过使用修饰的CPL1基因的遗传互补测定来确定，2）建立CPL1和HYL1之间的相互作用；确定CPL1-HYL1相互作用的模式并分析其与CPL1功能的关系；3)确定CPL1的RNA/基因沉默机制；将使用分子遗传分析和瞬时 RNAi 分析来分析 cpl1 突变对 DNA 甲基化、组蛋白修饰和小 RNA 积累/作用的影响。因此，该项目产生的信息主体将揭示植物基因调控的新机制，这也将对其他生物体产生影响。更广泛的影响该项目将由两个研究所的团队进行，该团队由两名博士后助理和一名研究生组成学生在本科生的支持下。 参与者将接受培训，了解和有效利用植物基因组信息和各种遗传资源，以及验证信息的分子生化技能。为了培养来自不同背景的未来科学家，生理学课程设有研究生水平的实验室模块。摩尔。植物应激反应基础课程将在 TAMU 教授，学生将获得实践经验，其中一些内容将在 UM 的类似课程中分享。 本课程已被许多田间农业领域的学生学习，因此，应提供可持续农业实践的基础知识。 本科生将使用 REU 补充剂参加研究项目。 与韩国庆尚国立大学启动并正在制定一项国际交流计划，访问学者和学生可以参与共同研究项目。 这是学生接触多学科和多元文化研究环境的绝佳机会。