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

植物基因表达是一个动态的过程，涉及多个层次的正调控和负调控。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，小RNA分子介导的RNA/基因沉默不仅可以在植物中引发转录和转录后基因沉默，也可以在其他真核生物中引发。小RNA生产的核心是含有双链RNA结合基序（DRM）的蛋白质。在真核生物中，植物含有独特的含有drm的蛋白质。羧基末端结构域（CTD）磷酸酶样1 （CPL1）蛋白包含一个n端磷酸酶结构域和串联重复的c端DRM结构域。本项目要测试的主要假设是植物特异性CPL蛋白微调了小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，我们的研究结果表明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进行基因沉默。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与HYL1之间的相互作用；确定CPL1- hyl1的相互作用模式，分析其与CPL1功能的关系；3)确定CPL1的RNA/基因沉默机制；cpl1突变对DNA甲基化、组蛋白修饰和小RNA积累/作用的影响将使用分子遗传学分析和瞬时RNAi分析进行分析。因此，在这个项目中产生的信息体将揭示植物基因调控的新机制，这也将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，该团队由两名博士后和一名研究生组成，并得到本科生的支持。与会者将接受培训，了解和有效利用植物基因组信息和各种遗传资源，以及分子生化技能，以验证信息。为了培养来自不同背景的未来科学家，物理学课程的研究生水平实验室模块。摩尔。植物胁迫反应的基础，学生将有实践经验，这将在TAMU教授，其中一些内容将在UM的类似课程中分享。这门课程已经被许多农田农业领域的学生学习过，因此，应该为可持续农业实践提供基础知识。本科生将使用REU补充剂参与研究项目。与韩国庆尚国立大学开展了国际交流项目，访问学者和学生可以相互参与研究项目。这是学生接触多学科、多文化研究环境的绝佳机会。

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.