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

转录和mRNA加工受RNA聚合酶II的羧基末端结构域（CTD）的磷酸化和去磷酸化调控，该结构域由YSPTSPS七肽的串联重复序列组成。先前的研究表明，植物CPL （CTD磷酸酶样）蛋白家族的成员在拟南芥中调节渗透胁迫和aba应答转录的差异。在拟南芥基因组中鉴定的CPL基因中，CPL1和CPL2代表了一种新的CTD磷酸酶，该酶在CTD重复序列中磷酸化Ser 5具有独特的位置特异性，并且含有双链RNA结合域。本项目旨在确定通过CTD磷酸化调控应激反应转录的分子网络。通过在CPL1零突变株（fry2-1）中表达突变后的CPL1，可以确定CPL1各结构域的体内功能。利用酵母双杂交系统鉴定与CPL1相互作用的蛋白（CIF: CPL1相互作用因子），并通过检测cpl和CIF突变等位基因的遗传相互作用进一步表征。我们将通过测试生理应激反应以及分析应激诱导的转录激活来分析CPL-CIF在应激反应中相互作用的功能。通过该项目，初级科学家（博士后、研究生/本科生）将接受培训，利用分子遗传和生化策略对核心转录机制进行综合研究。项目产生的数据/材料/突变体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.