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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，调节基因表达。 M6A谱的调节与肿瘤进展和化疗耐药有关。然而，目前仍不清楚m6A是如何参与这一过程的。了解潜在的机制是发现癌症治疗和诊断的新药物靶点和生物标记物的关键。最新研究已经指出了m6A在调节RNA引导的DNA修复中的新作用，这表明它可以介导癌症通过与DNA损伤和修复相互作用而发展。我们最近发现一架M6A位于 DNA聚合酶β转录本上的终止密码子被氧化β碱基损伤消除由环境毒物溴酸钾(KBrO_3)诱导，将其沉积转移到 POLβ的文字记录。我们假设m6A与RNA和DNA碱基损伤相互作用，以调节DNA修复。为了验证这一假设，我们将追求两个具体目标。目标1是确定m6A的分布和丰度可以受到癌细胞中RNA和DNA碱基损伤的调节。首先，我们将确定氧化型RNA和 DNA碱基损伤会破坏癌细胞中POLβ转录本上m6A的分布。这将在用KBrO_3处理HeLa细胞。我们将确定m6A谱是否可以被氧化的RNA碱基调节用DNARNA免疫沉淀法检测POLβ转录本上的损伤、8-oxoG和碱性位点(AP位点) (水滴)介导链断裂介导的RNA修饰图谱分析(水滴-SBRM)。然后我们将确定如果m6A谱可以通过DNA碱基损伤、8-oxodGs和Polβ模板上的AP位点来改变转录本使用滴滴介导链断裂介导的DNA修饰图谱分析(滴滴- SBDM)。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写入器和橡皮擦、METTL3/METTL14和FTO的亲和力。目标2是确定m6A是否可以改变 RNA碱基损伤的积累以调节癌细胞中的DNA修复。首先，我们将确定配置文件是否氧化损伤对POLβ转录本的影响可被m6A在METTL3下修饰 HeLa细胞缺乏症。M6A对小鼠肝细胞DNA和RNA氧化损伤的影响 KBrO_3将使用带有或不带有METTL3基因敲除的HeLa细胞来确定。M6A和M6A的分布 POLDNA转录本上的氧化碱基损伤与其模板β将是相关的。第二，我们将确定如果关键的BER酶的活性和底物结合亲和力可以在m6A和RNA中调节损伤位置依赖的方式。这项研究将证明表位转录修饰这一新概念与RNA和DNA碱基损伤相互作用，调节癌细胞中的DNA修复。结果将揭示一部小说 M6A与氧化RNA和DNA损伤相互作用以调节癌症背后的DNA修复的范式进步。因此，这项研究将有助于发现基于RNA的治疗、诊断和治疗的新靶点。环境致癌的预防。