Damaged RNA as a mediator of alkylation responses

项目摘要：本提案的目标是确定受损RNA在调节 癌症化疗期间的信号事件。在这笔赠款的前一个资金周期中，我们发现了一部小说 当细胞遇到烷化剂时，泛素依赖的信号通路特异性地启动，其中之一 癌症治疗中最常用的系统疗法。这一途径依赖于RNF113A E3 泛素连接酶，进而招募ASCC DNA解旋酶复合体以及ALKBH3 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 关于烷化损伤是如何在DNA上识别和修复的。然而，对RNA的损害远远超过 破坏任何遇到烷化剂的细胞中的DNA。使用新工具诱导或修复烷基化 RNA，我们发现烷基化的RNA是激活RNF113A的E3连接酶功能的必要条件和充分条件 并招募ASCC综合体。我们的证据还表明，RNF113A是一种磷酸化的E3连接酶， CDK12激酶负责其磷酸化。这些数据是我们模型的基础 这种RNA烷基化作用在激活E3连接酶(RNF113A)的激酶(CDK12)的上游，该连接酶在 转而招募一个烷基化修复复合体(ASCC-ALKBH3)。在这项建议中，我们将寻求了解 RNA烷基化重现RNF113A-ASCC-ALKBH3途径激活的机制基础 (目标1)。我们将表征这一途径在移除受损RNA、防止复制- 转录冲突和RNA-DNA杂交体(R-环)(目标2)。最后，我们将确定功能 CDK12介导的磷酸化在RNF113A激活中的后果及其在肿瘤中的作用 化疗反应(目标3)。值得注意的是，由于小分子CDK12抑制剂已经上市，我们将 能够测试以此通路为靶点是否会改善烷基化治疗反应。共同努力，我们的工作将 在核酸损伤信号中建立一个新的范式，其中RNA损伤在中介中发挥核心作用 DNA修复。

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.