Damaged RNA as a mediator of alkylation responses

受损的 RNA 作为烷基化反应的介质

• 批准号: 10608048
• 负责人: NIMA MOSAMMAPARAST
• 金额: $ 36.66万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10608048
• 关键词: Alkylating Agents; Alkylating Antineoplastic Agents; Alkylation; BRCA1 gene; Biochemical; Cell Nucleus; Cell Survival; Cells; Chemoresistance; Chemosensitization; Chemotherapy-Oncologic Procedure; Coal; Common Neoplasm; Complex; DNA; DNA Alkylation; DNA Damage; DNA Repair; Data; Dealkylation; Event; Evolution; Excision; Exposure to; Funding; Genetic Transcription; Genome; Glioblastoma; Grant; Human; Hybrids; Lesion; Maintenance; Malignant Neoplasms; Mediating; Mediator; Methyltransferase; Modality; Modeling; Nature; Nuclear; Nucleic Acids; Organism; Pathway interactions; Phosphorylation; Phosphotransferases; Play; RNA; RNA methylation; Repair Complex; Resistance; Role; Serinus; Signal Pathway; Signal Transduction; Testing; Therapeutic; Ubiquitin; Work; cancer therapy; chemotherapy; environmental agent; gene repression; genetic approach; genome integrity; helicase; improved; in vitro activity; inhibitor; innovation; kinase inhibitor; novel; overexpression; p53-binding protein 1; patient derived xenograft model; prevent; reconstitution; recruit; repaired; response; small molecule; small molecule inhibitor; tool; treatment response; tumor; ubiquitin-protein ligase; upstream kinase; virtual

Project Summary: The objective of this proposal is to define the role of damaged RNA in mediating signaling events during cancer chemotherapy. In the previous funding cycle of this grant, we uncovered a novel ubiquitin-dependent signaling pathway initiated specifically when cells encounter alkylating agents, one of the most commonly used systemic modalities for cancer treatment. This pathway depends on the RNF113A E3 ubiquitin ligase, which in turn recruits the ASCC DNA helicase complex as well as the ALKBH3 demethylase/dealkylase. To date, most of the studies on alkylation damage, including our own work, has focused on how alkylated lesions are recognized and repaired on DNA. Yet, damage to RNA is far more abundant than damaged DNA in any cell which encounters an alkylating agent. Using novel tools to induce or repair alkylated RNA, we have found that alkylated RNA is necessary and sufficient to activate the E3 ligase function of RNF113A and recruit the ASCC complex. Our evidence also indicates that RNF113A is a phospho-activated E3 ligase, and that the CDK12 kinase is responsible for its phosphorylation. This data serves as the basis for our model that RNA alkylation functions upstream of a kinase (CDK12) that activates an E3 ligase (RNF113A), which in turn recruits an alkylation repair complex (ASCC-ALKBH3). In this proposal, we will seek to understand the mechanistic basis for how RNA alkylation recapitulates the activation of the RNF113A-ASCC-ALKBH3 pathway (Aim 1). We will characterize the role of this pathway in removing damaged RNAs, preventing replication- transcription conflicts and RNA-DNA hybrids (R-loops) (Aim 2). Finally, we will determine the functional consequences of CDK12-mediated phosphorylation in the activation of RNF113A and its role in tumor chemotherapy responses (Aim 3). Notably, since small molecule CDK12 inhibitors are already available, we will be able to test whether targeting this pathway will improve alkylation therapy responses. Together, our work will establish a new paradigm in nucleic acid damage signaling, where RNA damage plays a central role in mediating DNA repair.

项目概述：本提案的目的是确定受损RNA在介导 癌症化疗期间的信号事件。在上一个资助周期中，我们发现了一本小说， 泛素依赖性信号通路在细胞遇到烷化剂时特异性启动， 最常用于癌症治疗的全身模式。该途径依赖于RNF 113 A E3 泛素连接酶，其又募集ASCC DNA解旋酶复合物以及ALKBH 3 脱甲基酶/脱烷基酶。到目前为止，大多数关于烷基化损伤的研究，包括我们自己的工作，都集中在 如何在DNA上识别和修复烷基化损伤。然而，对RNA的损伤远比 在任何细胞中遇到烷化剂的DNA受损。使用新工具诱导或修复烷基化 我们已经发现，烷基化的RNA是必需的，足以激活RNF 113 A的E3连接酶功能 招募ASCC复合体我们的证据还表明RNF 113 A是磷酸活化的E3连接酶， 并且CDK 12激酶负责其磷酸化。这些数据是我们模型的基础 RNA烷基化作用于激活E3连接酶（RNF 113 A）的激酶（CDK 12）的上游， turn募集烷基化修复复合物（ASCC-ALKBH 3）。在本提案中，我们将努力了解 RNA烷基化如何重现RNF 113 A-ASCC-ALKBH 3途径激活的机制基础 (Aim 1）。我们将描述这一途径在清除受损RNA，防止复制中的作用- 转录冲突和RNA-DNA杂合体（R环）（Aim 2）。最后，我们将确定函数 CDK 12介导的磷酸化在RNF 113 A活化中的作用及其在肿瘤中的作用 化疗反应（目的3）。值得注意的是，由于小分子CDK 12抑制剂已经可用，我们将 能够测试靶向该途径是否会改善烷基化治疗反应。我们一起努力， 在核酸损伤信号传导中建立了一个新的范例，其中RNA损伤在介导 DNA修复