Regulation of plant osmotic-stress-induced Gene Expression by Unique Ser 5-specific RNAP II CTD Phosphatases

RNA聚合酶II的羧基末端结构域(CTD)由YSPTSPS七肽的串联重复组成，转录和mRNA的加工是通过RNA聚合酶II的羧基末端结构域(CTD)的磷酸化和去磷酸化来调节的。以往的研究表明，植物CPL(CTD磷酸酶类似物)蛋白家族的成员在拟南芥中差异地调节渗透胁迫和ABA响应的转录。在拟南芥基因组中发现的CPL基因中，CPL1和CPL2是一种新的CTD磷酸酶，它对CTD重复序列中磷酸化的Ser5具有独特的位置特异性，并且含有双链RNA结合区。该项目旨在确定通过CTD磷酸化来调节应激反应转录的分子网络。CPL1每个结构域的体内功能将通过在CPL1缺失突变株(fry2-1)中表达诱变的CPL1来确定。与CPL1(CIF：Cp1-相互作用因子)相互作用的蛋白质将通过酵母双杂交系统进行鉴定，并将通过测试Cp1和CIF突变等位基因的遗传相互作用来进一步表征。CPL-CIF相互作用在应激反应中的功能将通过测试生理应激反应以及通过分析应激诱导的转录激活来分析。通过该项目，初级科学家(博士后、研究生/本科生)将接受使用分子遗传和生化策略对核心转录机制进行综合研究的培训。该项目产生的数据/材料/变种将在可用时存放在公共领域。

Transcription and mRNA processing are regulated by phosphorylation and dephosphorylation of the carboxyl-terminal domain (CTD) of RNA polymerase II, which consists of tandem repeats of a YSPTSPS heptapeptide. Previous studies showed that members of the plant CPL (CTD phosphatase-like) protein family differentially regulate osmotic-stress- and ABA-responsive transcription in Arabidopsis thaliana. Among the CPL genes identified in the Arabidopsis genome, CPL1 and CPL2 represent a novel CTD phosphatase, which exhibits unique position specificity for phosphorylated Ser 5 in the CTD repeat, and contains double-stranded RNA binding domains. This project aims to identify the molecular network that regulates stress-responsive transcription through CTD phosphorylation. The in vivo function of each domain of CPL1 will be determined by expressing mutagenized CPL1 in a CPL1 null mutant strain (fry2-1). Proteins interacting with CPL1 (CIF: CPL-interacting factor) will be identified using the yeast two-hybrid system, and will be further characterized by testing genetic interactions of cpl and cif mutant alleles. Functionality of CPL-CIF interaction during stress response will be analyzed by testing physiological stress responses, as well by analyzing stress-inducible transcriptional activation. Through the project, junior scientists (postdocs, graduate/undergraduate students) will be trained for integrated studies of core transcriptional machinery using molecular genetic and biochemical strategies. The data/materials/mutants generated by the project will be deposited in the public domain as they become available.