Determining the role of RNA abasic sites in gene regulation

确定 RNA 无碱基位点在基因调控中的作用

• 批准号: 10572004
• 负责人: Vivian G Cheung
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-06 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10572004
• 关键词: Adenosine; Apolipoprotein E; Base Excision Repairs; Binding Proteins; Cells; DNA; DNA Damage; DNA Polymerase II; DNA Repair; Data; Endonuclease I; Enhancers; Enzymes; Gene Expression; Gene Expression Regulation; Genetic Transcription; Hybrids; Length; Mass Spectrum Analysis; Mediating; Methylation; Modification; Nucleic Acids; Pathway interactions; Process; Proteins; Publishing; RNA; RNA Polymerase II; RNA Precursors; Reaction; Regulation; Research; Ribosomal RNA; Ricin; Role; Signal Transduction; Site; Stress; Structure; Untranslated RNA; base; cell type; endonuclease; genome-wide; hammerhead ribozyme; methylpurine; nucleic acid structure; plant poison; prevent; response

RNA abasic sites are recently identified modifications in RNA that are generated by methylpurine glycosylase to regulate gene expression. They are abundant in RNA and they stabilize RNA yet we do not know much about how they form, their precursors, and their functions. DNA abasic sites were identified in the 1960s and those discoveries led to the elucidation of the base excision repair pathway in DNA damage. But RNA abasic sites were largely unknown, except those generated by the plant poison, ricin, in ribosomal RNA. While studying proteins that bind to the nucleic acid structure, R-loop, we identified RNA modification enzymes and methylpurine glycosylase as well as apurinic/apyrimidinic endonuclease I that were known to process DNA abasic sites. We have now shown that methylpurine glycosylase cleaves the glycosidic bond in RNA to form abasic sites and these reactions occur on RNA in RNA/DNA hybrids of R-loops. By mass spectrometry, we found that RNA abasic sites are rather abundant, there are tens to hundreds of thousands of them in each cell. We also found in an enhancer RNA of APOE, N6- methyladenosines are cleaved by methylpurine glycosylase to form RNA abasic sites on R- loops. The abasic sites then stabilize R-loops and pause RNA Polymerase II transcription. We show that this mechanism keeps the noncoding RNA poised to activate APOE expression in response to cellular demands. Given that we have early evidence for the role of RNA abasic sites in the regulation of noncoding RNA, it is necessary to better understand them. We propose to 1) examine the genome-wide effect of nucleic-acid-mediated pausing on noncoding RNA, 2) identify other glycosylases that form RNA abasic sites, and 3) determine what other modified RNA bases are precursors of RNA abasic sites. Together, these results will characterize RNA abasic sites by finding how they form and what they do.

RNA 无碱基位点是最近发现的 RNA 修饰，由 甲基嘌呤糖基化酶调节基因表达。它们富含 RNA，并且 稳定RNA，但我们对它们的形成方式、它们的前体以及它们的作用知之甚少。 功能。 DNA脱碱基位点在20世纪60年代被鉴定出来，这些发现导致了对DNA脱碱基位点的阐明。 DNA损伤中的碱基切除修复途径。但 RNA 脱碱基位点很大程度上是未知的， 除了核糖体 RNA 中由植物毒物蓖麻毒素产生的那些。在研究蛋白质时 与核酸结构 R 环结合，我们鉴定了 RNA 修饰酶和 甲基嘌呤糖基化酶以及无嘌呤/无嘧啶核酸内切酶 I 已知 处理DNA脱碱基位点。 我们现在已经证明，甲基嘌呤糖基化酶会裂解 RNA 中的糖苷键，形成 无碱基位点和这些反应发生在 R 环的 RNA/DNA 杂交体中的 RNA 上。按质量计 光谱分析，我们发现RNA脱碱基位点相当丰富，有几十到几百个 每个牢房里有数千个。我们还在 APOE 的增强子 RNA 中发现了 N6- 甲基腺苷被甲基嘌呤糖基酶切割，在 R- 上形成 RNA 无碱基位点 循环。然后，脱碱基位点稳定 R 环并暂停 RNA 聚合酶 II 转录。我们 表明这种机制使非编码 RNA 能够激活 APOE 表达 对细胞需求的响应。 鉴于我们有早期证据证明 RNA 无碱基位点在调节中的作用 非编码RNA，有必要更好地了解它们。我们建议 1) 检查 核酸介导的暂停对非编码 RNA 的全基因组影响，2) 确定其他 形成 RNA 无碱基位点的糖基化酶，以及 3) 确定其他修饰的 RNA 碱基是什么 RNA脱碱基位点的前体。总之，这些结果将通过以下方式表征 RNA 无碱基位点： 找出它们是如何形成的以及它们的作用。