Regulation of RNA polymerase II transcription in trypanosomatids

锥虫中 RNA 聚合酶 II 转录的调控

• 批准号: 9789811
• 负责人: ROBERT S SABATINI
• 金额: $ 43.79万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-06 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789811
• 关键词: Address; African Trypanosomiasis; Antiparasitic Agents; Area; Binding; Binding Proteins; Biological Models; Biology; C-terminal; Cells; Chromatin; Co-Immunoprecipitations; Code; Communicable Diseases; Complex; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Development; Diagnosis; Disease; Dissociation; ENG gene; Environment; Epigenetic Process; Eukaryota; Evolution; Gene Cluster; Gene Expression; Gene Expression Regulation; Gene Silencing; Gene Structure; Genes; Genetic Transcription; Goals; Grant; Histone H3; Human; In Vitro; Individual; Infection; Intervention; Lead; Leishmania; Link; Mediating; Modeling; Modification; Morbidity - disease rate; Nuclear; Operon; Parasites; Pathway interactions; Poly A; Polyadenylation; Prevention approach; Process; Protein Dephosphorylation; Protein phosphatase; Proteins; Protozoa; Public Health; RNA; RNA-Binding Proteins; Regulation; Research; Role; Running; Sedimentation process; Site; Structure; Technology; Testing; Thymine; Torpedo; Transcriptional Regulation; Trypanosoma; Trypanosoma brucei brucei; Variant; Vector-transmitted infectious disease; Work; base; epigenetic regulation; genetic regulatory protein; genome-wide; helicase; in vivo; insight; mortality; neglect; next generation sequencing; novel; novel strategies; pathogen; premature; prevent; recruit; spleen exonuclease; transcription termination

Project Summary Trypanosoma brucei is a single cell protozoan parasite that causes human African Sleeping Sickness, that is fatal if untreated. Related trypanosomatid pathogens, including numerous Leishmania species, cause even more morbidity and mortality worldwide. The regulation of gene expression is essential for parasite development and survival in their sequential host environments. Exactly how trypanosomatid gene expression is regulated is currently not well understood, but the relative contribution of transcriptional control was thought to be absent. We have demonstrated a role for a modified DNA base, called base J, and histone H3 variant (H3.V) in the regulation of RNA polymerase II (Pol II) transcription termination in T. brucei and L. major. This includes sites within polycistronic gene clusters leading to “premature” termination and silencing of genes downstream. Our long-term goal is to dissect epigenetic mechanisms of gene expression in trypanosomatids. The objective of this grant is to determine the mechanism of J/H3.V regulated Pol II transcription termination in T. brucei. The central hypothesis is that base J/H3V regulates termination via regulating specific stages of a “torpedo-PP1” termination mechanism. The torpedo component involves 5'-3' exonuclease (XRND), helicase (Sen1) and an RNA binding protein (RBP33). Pol II pausing and stimulation of the torpedo mechanism in other eukayotes involves dephosphorylation of the C-terminal domain of Pol II by PP1 protein phosphatase as part of the multimeric PTW/PP1 complex composed of regulatory subunits PNUTS, Tox4, and Wdr82 and PP1. We have identified a similar complex in kinetoplastids, called PJJW/PP1, that replaced Tox4 with a J-binding protein (JBP3). These data led to the current model where base J (via JBP3) recruits the PJJW/PP1 complex at termination sites and PP1 dephosphorylates Pol II CTD, pausing elongation allowing the torpedo mechanism to efficiently dissociate Pol II. Our specific aims will test the following hypothesis: Aim 1 will directly test our torpedo model by assessing the role of these protein factors in transcription termination in T. brucei. We will then analyze the function of individual protein components using a combination of in vitro and in vivo analyses. Aim 2 will characterize the PJJW/PP1 complex, including the roles of PNUTS and JBP3 as the assembly and chromatin recruitment factor, respectively. Aim 3 will elucidate the role of the PJJW/PP1 complex in termination, including the function of PP1 in modifying the CTD of pol II. The proposed work will provide a fundamental understanding trypanosomatid gene expression, yielding a comprehensive view of the role of transcriptional control and clues to it mechanisms. These studies may prove useful in identifying novel approaches to prevention, treatment and diagnosis of the debilitating and deadly diseases caused by these parasites. ! ! ! !

项目概要 布氏锥虫是一种单细胞原生动物寄生虫，会导致人类非洲昏睡病，即 如果不治疗，相关的锥虫病原体，包括许多利什曼原虫，甚至会导致致命。 全球发病率和死亡率更高 基因表达的调控对于寄生虫至关重要。 锥虫基因在其连续宿主环境中的发育和生存到底是如何的。 目前尚不清楚转录控制的相对贡献 我们已经证明了修饰的 DNA 碱基（称为碱基 J）和组蛋白 H3 变体的作用。 (H3.V) 在 T. brucei 和 L. Major 中对 RNA 聚合酶 II (Pol II) 转录终止的调节。 包括多顺反子基因簇内导致基因“过早”终止和沉默的位点 我们的长期目标是剖析锥虫基因表达的表观遗传机制。 该资助的目的是确定 J/H3.V 调节 Pol II 转录终止的机制 T. brucei 的中心假设是基础 J/H3V 通过调节 a 的特定阶段来调节终止。 “torpedo-PP1”终止机制涉及5'-3'核酸外切酶（XRND）、解旋酶。 (Sen1) 和 RNA 结合蛋白 (RBP33) 暂停和刺激其他鱼雷机制。 真核生物涉及 PP1 蛋白磷酸酶对 Pol II C 末端结构域的去磷酸化，作为 由调节亚基 PNUTS、Tox4、Wdr82 和 PP1 We 组成的多聚体 PTW/PP1 复合物。 已在动质体中鉴定出一种类似的复合物，称为 PJJW/PP1，它用 J 结合取代了 Tox4 这些数据得出了当前的模型，其中碱基 J（通过 JBP3）招募 PJJW/PP1 复合物。 在终止位点，PP1 使 Pol II CTD 去磷酸化，暂停延伸，允许鱼雷 有效解离 Pol II 的机制我们的具体目标将检验以下假设：目标 1 将直接 通过评估这些蛋白质因子在 T. brucei 转录终止中的作用来测试我们的鱼雷模型。 然后，我们将结合体外和体内分析单个蛋白质成分的功能 目标 2 将描述 PJJW/PP1 复合体的特征，包括 PNUTS 和 JBP3 的作用。 目标 3 将分别阐明 PJJW/PP1 的作用。 终止的复杂性，包括 PP1 在修改 pol II 的 CTD 中的功能。 提供对锥虫基因表达的基本了解，从而全面了解锥虫基因表达 转录控制的作用及其机制的线索这些研究可能有助于识别新的机制。 预防、治疗和诊断由这些疾病引起的衰弱和致命疾病的方法 寄生虫。 ！ ！ ！ ！