The role of N6-methyladenosine RNA modification in programmed and aberrant DNA mutagenesis in B cells

N6-甲基腺苷 RNA 修饰在 B 细胞程序性和异常 DNA 突变中的作用

• 批准号: 10259665
• 负责人: Uttiya Basu
• 金额: $ 51.19万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-09 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10259665
• 关键词: 3-Dimensional; Alleles; Antibodies; Antibody Affinity; Antigens; Automobile Driving; B lymphoid malignancy; B-Cell Development; B-Lymphocytes; Biochemistry; Biological Assay; Biology; Bone Marrow; Cell Line; Cell Lineage; Cell physiology; Chromosomal Instability; Chromosome abnormality; Code; Complex; Correlative Study; Coupled; DNA; DNA Double Strand Break; DNA Probes; DNA Sequence Alteration; DNA Single Strand Break; DNA Structure; Data; Defect; Disease; Enzymes; Evaluation; Event; Frequencies; Gene Activation; Gene Expression; Gene Rearrangement; Generations; Genes; Genetic Recombination; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Heavy-Chain Immunoglobulins; Hematopoiesis; Heterodimerization; IgG1; Immune System Diseases; Immunize; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Laboratories; Light; Lymphoid; Lymphomagenesis; Mature B-Lymphocyte; Mediating; Methylation; Microscopy; Modification; Molecular; Multiple Myeloma; Mus; Mutagenesis; Mutation; Pathway interactions; Peyer' s Patches; Play; Post-Transcriptional Regulation; Prevention; Process; Production; Property; Proteins; RNA; RNA Degradation; RNA Processing; RNA methylation; Research Design; Resolution; Rest; Role; Single-Stranded DNA; Site; Somatic Mutation; Sterility; Structure; Structure of germinal center of lymph node; System; Transcript; Untranslated RNA; V(D)J Recombination; activation-induced cytidine deaminase; adaptive immune response; cancer cell; cellular development; ds-DNA; epigenomics; exosome; experimental study; genome integrity; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; leukemia/lymphoma; mouse genetics; mouse model; mutant; prevent; progenitor; recruit; single molecule; transcriptome sequencing

PROJECT SUMMARY Background: VDJ recombination, Class switch recombination (CSR) and somatic hypermutation (SHM) are three B lymphocyte specific processes that mediate antibody gene diversification. VDJ recombination requires the DNA double strand generation by the Recombination activation genes (RAG1 an RAG2) where as CSR and SHM requires the single-strand DNA break activity of the Activation Induced Deaminase (AID) enzyme. Both RAG1/2 and AID activities are coupled with noncoding RNA transcription at sites of DNA break/mutation. The properties of the ncRNAs generated at sites of programmed DNA breaks are poorly characterized in B cells. Recently advances in biology has provided compelling evidence that post-transcriptional and co-transcriptional modification of ncRNAs determine a component of RNA epigenomics and have significant role in driving cellular development and function. In this application, supported by preliminary data generated in our laboratory, we are evaluating the role of RNA modification N6-methyladenosine (m6A) and its associated enzymes METTL3 and METTL14 in B cell development, function and genomic integrity. Objective/Hypothesis: In his proposal, we will determine how RNA methylation m6A on transcripts generated in the IgH locus and the rest of the B cell genome controls programmed DNA recombination, antibody gene diversification and prevents chromosomal instability. Specific Aims: Aim 1: Aim 1: Are m6A modifying enzymes Mettl3 and Mettl14 important for class switch recombination, somatic hypermutation, and/or for preventing genomic stability in B cells? ; AIM 2: What is the mechanism by which m6A modification promotes IgH DNA recombination and/or prevents genomic instability? AIM 3: Do RNA methylation and coupled RNA surveillance pathways have a role in early B cell development and during VDJ recombination? Study Design: Using cell lines and mouse models, we will study mechanism by which RNA m6A methylation plays a role in programmed DNA recombination and protection of B cell genomic integrity. We will evaluate the mechanism of formation of single-strand DNA structures formation and RNA surveillance at sites of AID activity in matured B cells and the mechanism by which inhibitory RNAs are degraded at sites of RAG activity in B cells during VDJ recombination. We will also evaluate the molecular mechanism by which RNA modification promotes programmed DNA rearrangements. We use a combination of mouse genetics, genomics, biochemistry and 3D-STORM microscopy to accomplish the goals of the proposed project. Disease Relevance: B cells are a central component of the adaptive immune response, but also prone to undergo leukemias and lymphomas when antibody gene diversification processes are not well controlled. Proposed studies leads to a better understanding of the mechanism of antibody gene diversification but also educate us how B cell cancer (specially in context of DLBCL and Multiple Myeloma) are prevented.

项目概要 背景：VDJ重组、类别转换重组（CSR）和体细胞超突变（SHM）是 介导抗体基因多样化的三个 B 淋巴细胞特异性过程。 VDJ重组需要 由重组激活基因（RAG1 和 RAG2）产生 DNA 双链，其中 CSR 和 SHM 需要激活诱导脱氨酶 (AID) 的单链 DNA 断裂活性。两个都 RAG1/2 和 AID 活性与 DNA 断裂/突变位点的非编码 RNA 转录相结合。这 在 B 细胞中，在程序性 DNA 断裂位点生成的 ncRNA 的特性尚不清楚。 最近生物学的进展提供了令人信服的证据，证明转录后和共转录 ncRNA 的修饰决定了 RNA 表观基因组学的一个组成部分，并在驱动 细胞的发育和功能。在此应用程序中，由我们生成的初步数据支持 实验室，我们正在评估 RNA 修饰 N6-甲基腺苷 (m6A) 及其相关的作用 METTL3 和 METTL14 酶在 B 细胞发育、功能和基因组完整性中的作用。 目标/假设：在他的提议中，我们将确定转录本上的 RNA 甲基化 m6A 如何 在 IgH 基因座中产生，B 细胞基因组的其余部分控制程序性 DNA 重组， 抗体基因多样化并防止染色体不稳定。 具体目标： 目标 1： 目标 1：m6A 修饰酶 Mettl3 和 Mettl14 对于类别转换是否重要 重组、体细胞超突变和/或防止 B 细胞基因组稳定性？ ;目标 2：什么是 m6A 修饰促进 IgH DNA 重组和/或阻止基因组重组的机制 不稳定？目标 3：RNA 甲基化和偶联 RNA 监视途径在早期 B 细胞中起作用吗 发展和VDJ重组期间？ 研究设计：使用细胞系和小鼠模型，我们将研究 RNA m6A 甲基化的机制 在程序性 DNA 重组和保护 B 细胞基因组完整性中发挥作用。我们将评估 AID位点单链DNA结构形成和RNA监测的形成机制 成熟 B 细胞中的活性以及抑制性 RNA 在 RAG 活性位点被降解的机制 VDJ 重组期间 B 细胞中。我们还将评估 RNA 的分子机制 修饰促进程序性 DNA 重排。我们结合小鼠遗传学、基因组学、 生物化学和 3D-STORM 显微镜来实现拟议项目的目标。 疾病相关性：B 细胞是适应性免疫反应的核心组成部分，但也容易发生 当抗体基因多样化过程得不到很好控制时，就会患上白血病和淋巴瘤。 拟议的研究可以更好地理解抗体基因多样化的机制，但也 告诉我们如何预防 B 细胞癌（特别是 DLBCL 和多发性骨髓瘤）。