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

植物基因表达是一个动态过程，涉及多个水平的正调控和负调控。 最近的研究强调了由小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还可以在其他真核生物中触发转录和转录后基因沉默。 小RNA产生的核心是含有双链（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（CPL 1）蛋白含有N-末端磷酸酶结构域和串联重复的C-末端DRM结构域。在这个项目中要测试的主要假设是，植物特异性CPL蛋白微调小RNA介导的基因沉默的水平。 由于CTD磷酸化可以调节pol II复合物的活性，我们的研究结果意味着植物进化了一种额外的基因表达微调机制，该机制使用转录延伸的成分进行基因沉默。 本项目将通过确定CPL 1在基因沉默中的作用来剖析转录延伸机制和基因沉默之间的相互作用。 本项目的具体目标是：1）建立CPL 1在体内功能所必需的结构：CPL 1蛋白的必需结构域将通过使用修饰的CPL 1基因的遗传互补测定来确定; 2）建立CPL 1和HYL 1之间的相互作用;确定CPL 1-HYL 1相互作用的模式并分析其与CPL 1功能的关系，3）确定CPL 1的RNA/基因沉默机制;将使用分子遗传学分析以及瞬时RNAi分析来分析CPL 1突变对DNA甲基化、组蛋白修饰和小RNA积累/作用的影响。因此，本项目所产生的信息将揭示植物基因调控的新机制，并对其他生物产生影响。更广泛的影响本项目将由两个研究所组成的团队进行，包括两名博士后和一名研究生，并得到本科生的支持。 参加者将接受培训，以了解和有效利用植物基因组信息和各种遗传资源，以及分子生物化学技能，以验证信息。为了培养来自不同背景的未来科学家，生理学课程的研究生水平实验室模块。植物胁迫反应的基础，学生将有实践经验，将在TAMU教授，其中一些组成部分将在UM的类似课程中分享。 本课程已采取了许多学生在外地为导向的农业领域，因此，应提供可持续农业实践的基础知识。 本科生将参加使用REU补充研究计划。 已经启动了一个国际交流计划，并正在与庆尚国立大学、韩国韩国，访问学者和学生可以参加相互研究项目。 这是一个很好的机会，让学生接触到多学科和多元文化的研究环境。

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.