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: 10721410
• 负责人: Uttiya Basu
• 金额: $ 5.31万
• 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/10721410
• 关键词: 3-Dimensional; Antibodies; Antibody Affinity; Antigens; Automobile Driving; B lymphoid malignancy; B-Cell Development; B-Lymphocytes; Biochemistry; Biological Assay; Biology; Bone Marrow; CD19 gene; Cell Line; Cell Lineage; Cell physiology; Chromatin; Chromosomal Instability; Chromosome abnormality; Code; Complex; Correlative Study; Coupled; DNA; DNA Double Strand Break; 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; IGH@ gene cluster; IgG1; Immune System Diseases; Immunize; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulins; Laboratories; Light-Chain Immunoglobulins; LoxP-flanked allele; Lymphoid; Lymphomagenesis; Mature B-Lymphocyte; Mediating; Methylation; Microscopy; Modification; Molecular; Multiple Myeloma; Mus; Mutagenesis; Mutation; Pathway interactions; Peyer' s Patches; Play; Post-Transcriptional RNA Processing; Post-Transcriptional Regulation; Prevention; Process; Production; Property; Proteins; RNA; RNA Degradation; RNA Processing; RNA methylation; Rag1 Mouse; 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; humoral immunity deficiency; in vivo; large cell Diffuse non-Hodgkin' s lymphoma; leukemia/lymphoma; mouse genetics; mouse model; mutant; posttranscriptional; prevent; progenitor; recruit; single molecule; superresolution microscopy; 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.

项目摘要 背景：VDJ重组，类开关重组（CSR）和躯体超成名（SHM）为 介导抗体基因多样化的三个B淋巴细胞特定过程。 VDJ重组需要 通过重组激活基因（rag1 a rag2）的DNA双链生成，其中为CSR和 SHM需要激活诱导的脱氨酶（AID）酶的单链DNA断裂活性。两个都 RAG1/2和AID活性与DNA断裂/突变部位的非编码RNA转录结合。这 在编程DNA断裂部位产生的NCRNA的性质在B细胞中的特征很差。 最近的生物学进展提供了令人信服的证据，表明后转录和共同转录 NCRNA的修饰决定了RNA表观基因组学的成分，并且在驱动中具有重要作用 细胞发育和功能。在此应用程序中，由我们在我们的初步数据支持 实验室，我们正在评估RNA修饰N6-甲基读腺苷（M6A）及其相关的作用 B细胞发育，功能和基因组完整性中的METTL3和METTL14酶。 客观/假设：在他的提议中，我们将确定RNA甲基化M6A如何在转录本上 在IGH基因座和B细胞基因组的其余部分中产生的DNA重组， 抗体基因多样化并防止染色体不稳定性。 具体目的：目标1：目标1：M6A修改酶Mettl3和Mettl14对于类开关很重要 重组，体细胞超数和/或用于预防B细胞中基因组稳定性？ ;目标2：什么是 M6A修饰促进IGH DNA重组和/或防止基因组的机制 不稳定？ AIM 3：RNA甲基化和耦合RNA监视途径在早期B细胞中起作用 开发和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 在编程的DNA重组和B细胞基因组完整性的保护中起作用。我们将评估 在AID部位形成单链DNA结构形成和RNA监测的机理 成熟的B细胞的活性以及在破布活性部位降解抑制性RNA的机制 在VDJ重组期间B细胞中。我们还将评估RNA的分子机制 修改促进编程的DNA重排。我们结合了小鼠遗传学，基因组学， 生物化学和3D-STORM显微镜以实现拟议项目的目标。 疾病相关性：B细胞是自适应免疫反应的核心组成部分，但也容易 当抗体基因多样化过程无法很好地控制时，会经历白血病和淋巴瘤。 拟议的研究会更好地理解抗体基因多样性的机制，但也可以更好地理解 教育我们如何阻止B细胞癌（特别是在DLBCL和多发性骨髓瘤的背景下）。