N6-methyladenosine (m6A) Interplays with RNA and DNA Damage to Regulate DNA Repair

N6-甲基腺苷 (m6A) 与 RNA 和 DNA 损伤相互作用以调节 DNA 修复

基本信息

批准号：
10649063
负责人：
Yuan Liu
金额：
$ 7.38万
依托单位：
FLORIDA INTERNATIONAL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-03-07 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10649063
关键词：
8-hydroxyguanosine Address Affinity BRCA1 gene Base Excision Repairs Binding Biological Assay Cell physiology Cells Complex DNA DNA Damage DNA Modification Process DNA Polymerase beta DNA Repair DNA glycosylase DNA-Directed DNA Polymerase Deposition Development Diagnosis Disease Drug resistance Environment Environmental Carcinogens Excision Gel Gene Expression Genes Guide RNA Hela Cells Homologous Gene Human Hybrids Immunoprecipitation Knock-out Knowledge Laboratories Lesion Location Malignant Neoplasms Maps Measures Mediating Messenger RNA Methods Methylation Methyltransferase Modification Outcome Play Prevention Process RNA Regulation Resolution Role Site Terminator Codon Testing Therapeutic Toxic Environmental Substances Transcript base cancer biomarkers cancer cell cancer diagnosis cancer drug resistance cancer prevention cancer therapy carcinogenesis demethylation environmental carcinogenesis epitranscriptomics fat mass and obesity-associated protein insight invention knock-down new therapeutic target novel potassium bromate public health relevance recruit repair enzyme response tumor progression

项目摘要

N6-methyladenosine (m6A) is the most common modification in mRNA and regulates gene expression. Modulation of m6A profiles is associated with cancer progression and chemotherapeutic drug resistance. However, it remains unknown how m6A is involved in the processes. Understanding the underlying mechanisms is the key to the discovery of new drug targets and biomarkers for cancer therapy and diagnosis. Recent studies have pointed to a new role of m6A in regulating RNA-guided DNA repair suggesting that it can mediate cancer development by interplaying with DNA damage and repair. We recently found that an m6A located next to the stop codon on the transcript of DNA polymerase β (Pol β) was eliminated by oxidative RNA base damage induced by the environmental toxicant, potassium bromate (KBrO3), shifting its deposition to the upstream of the Pol β transcript. We hypothesize that m6A interplays with RNA and DNA base damage to regulate DNA repair. To test this hypothesis, we will pursue two Specific Aims. Aim 1 is to determine if m6A profiles and abundance can be modulated by RNA and DNA base damage in cancer cells. First, we will determine if oxidative RNA and DNA base damage can disrupt the profiles of m6A on the Pol β transcript in cancer cells. This will be tested in HeLa cells treated with KBrO3. We will determine if m6A profiles can be modulated by oxidative RNA base damage, 8-oxoG, and abasic sites (AP sites) on the Pol β transcript using DNA-RNA immunoprecipitation (DRIP)-mediated strand break-mediated RNA modification profiling assay (DRIP-SBRM). We will then determine if the m6A profiles can be altered by DNA base damage, 8-oxodGs, and AP sites on the DNA template of Pol β transcript using the DRIP-mediated strand break-mediated DNA modification profiling assay (DRIP- SBDM). Second, we will determine if RNA and DNA base damage can alter the activity and substrate binding affinity of the m6A writer and eraser, METTL3/METTL14 and FTO. Aim 2 is to determine if m6A can alter the accumulation of RNA base damage to modulate DNA repair in cancer cells. First, we will determine if the profiles of oxidative DNA and RNA base damage on the Pol β transcript can be modified by m6A under METTL3 deficiency in HeLa cells. The effects of m6A on the profiles of oxidative DNA and RNA base damage induced by KBrO3 will be determined using HeLa cells with or without METTL3 gene knockdown. The profiles of m6A and oxidative base damage on the Pol β transcript and its template DNA will be correlated. Second, we will determine if the activity and substrate binding affinity of the key BER enzymes can be modulated in an m6A and RNA damage location-dependent manner. The study will prove the new concept that epitranscriptomic modifications interplay with RNA and DNA base damage to regulate DNA repair in cancer cells. The results will reveal a novel paradigm for m6A to interplay with oxidative RNA and DNA damage to modulate DNA repair that underlies cancer progression. Thus, the study will facilitate the discovery of new targets for RNA-based treatment, diagnosis, and prevention of environmentally-induced cancer.

N6-甲基腺苷 (m6A) 是 mRNA 中最常见的修饰并调节基因表达。 m6A 谱的调节与癌症进展和化疗药物耐药性相关。然而，目前尚不清楚 m6A 如何参与这些过程。是发现癌症治疗和诊断的新药物靶点和生物标志物的关键。指出 m6A 在调节 RNA 引导的 DNA 修复中的新作用，表明它可以介导癌症我们最近发现 m6A 位于 DNA 损伤和修复的旁边。 DNA 聚合酶 β (Pol β) 转录本上的终止密码子因 RNA 碱基氧化损伤而被消除由环境毒物溴酸钾（KBrO3）引起，将其沉积转移到上游我们研究了 m6A 与 RNA 和 DNA 碱基损伤相互作用来调节 DNA 修复。为了检验这一假设，我们将追求两个具体目标 1 是确定 m6A 概况和丰度。首先，我们将确定氧化性RNA是否可以通过癌细胞中的RNA和DNA碱基损伤来调节。 DNA 碱基损伤会破坏癌细胞中 Pol β 转录本上的 m6A 特征，这将在细胞中进行测试。用 KBrO3 处理的 HeLa 细胞将确定 m6A 谱是否可以通过氧化 RNA 碱基进行调节。使用 DNA-RNA 免疫沉淀法检测 Pol β 转录本上的损伤、8-oxoG 和脱碱基位点（AP 位点）然后我们将确定 (DRIP) 介导的链断裂介导的 RNA 修饰分析 (DRIP-SBRM) 测定。 Pol β DNA 模板上的 DNA 碱基损伤、8-oxodG 和 AP 位点是否可以改变 m6A 谱使用 DRIP 介导的链断裂介导的 DNA 修饰分析测定（DRIP- 其次，我们将确定 RNA 和 DNA 碱基损伤是否会改变活性和底物结合。 m6A 写入器和擦除器、METTL3/METTL14 和 FTO 的亲和力目标 2 是确定 m6A 是否可以改变 RNA 碱基损伤的积累来调节癌细胞中的 DNA 修复首先，我们将确定这些特征是否存在。 METTL3下m6A可以修饰氧化DNA和RNA碱基对Pol β转录物的损伤 m6A 缺陷对 HeLa 细胞中氧化 DNA 和 RNA 碱基损伤的影响。 KBrO3 将使用具有或不具有 METTL3 基因敲低的 HeLa 细胞测定 m6A 和 m6A 的概况。其次，我们将确定 Pol β 转录本及其模板 DNA 上的氧化碱基损伤是否相关。关键 BER 酶的活性和底物结合亲和力是否可以在 m6A 和 RNA 中调节该研究将证明表观转录组修饰的新概念。与RNA和DNA碱基损伤相互作用来调节癌细胞中的DNA修复，这一结果将揭示一种新的机制。 m6A 与氧化 RNA 和 DNA 损伤相互作用以调节癌症基础 DNA 修复的范例因此，该研究将有助于发现基于 RNA 的治疗、诊断和进展的新靶点。预防环境诱发的癌症。