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无碱基位点是最近鉴定的RNA中的修饰，其通过以下方式产生： 甲基嘌呤糖基化酶调节基因表达。它们富含RNA， 稳定RNA，但我们对它们如何形成、它们的前体和它们的 功能协调发展的 DNA无碱基位点在20世纪60年代被鉴定，这些发现导致了对DNA序列的阐明。 DNA损伤的碱基切除修复途径。但是RNA的无碱基位点在很大程度上是未知的， 除了那些由植物毒素蓖麻毒素在核糖体RNA中产生的。在研究蛋白质时 结合到核酸结构，R环，我们确定了RNA修饰酶， 甲基嘌呤糖基化酶以及脱嘌呤/脱嘧啶核酸内切酶I，它们已知 处理DNA无碱基位点。 我们现在已经证明，甲基嘌呤糖基化酶切割RNA中的糖苷键， 无碱基位点，这些反应发生在R环的RNA/DNA杂合体中的RNA上。质量 光谱，我们发现RNA的脱碱基位点相当丰富，有几十到几百个， 每一个细胞里都有成千上万个。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化酶切割，在R- 循环脱碱基位点然后稳定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处于激活APOE表达的状态， 对细胞需求的反应。 鉴于我们有早期的证据表明RNA脱碱基位点在调节细胞凋亡中的作用， 非编码RNA，有必要更好地了解它们。我们建议1）研究 核酸介导的暂停对非编码RNA的全基因组效应，2）鉴定其他 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无碱基位点的前体。总之，这些结果将通过以下方式表征RNA无碱基位点： 发现它们是如何形成的以及它们的作用。