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

项目摘要 背景：VDJ重组、类别转换重组（CSR）和体细胞超突变（SHM）是近年来发现的一种基因突变， 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 通过扩增激活基因（RAG 1和RAG 2）产生DNA双链，其中作为CSR， SHM需要活化诱导脱氨酶（AID）的单链DNA断裂活性。两 RAG 1/2和AID活性在DNA断裂/突变位点与非编码RNA转录偶联。的 在程序性DNA断裂位点产生的ncRNA的性质在B细胞中表征不佳。 生物学的最新进展提供了令人信服的证据，即转录后和共转录 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 细胞发育和功能。在这个应用程序中，由我们的初步数据支持， 在实验室，我们正在评估RNA修饰N6-甲基腺苷（m6 A）及其相关的作用， 酶L3和酶L14在B细胞发育、功能和基因组完整性中的作用。 目的/假设：在他的提议中，我们将确定转录本上的RNA甲基化m6 A是如何发生的。 产生于IgH基因座和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，防止染色体不稳定。 具体目的：目的1：目的1：m6 A修饰酶Mettl 3和Mettl 14对于类别转换是否重要 重组、体细胞超突变和/或防止B细胞中的基因组稳定性？目标2：什么是 m6 A修饰促进IgH DNA重组和/或阻止基因组DNA重组的机制 不稳定？目的3：RNA甲基化及其相关的RNA监视通路在早期B细胞中是否起作用 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 m6 A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起作用。我们将评估 艾滋病病毒感染部位单链DNA结构形成和RNA监测机制 在成熟B细胞中的活性以及抑制性RNA在RAG活性位点降解的机制 在B细胞中的VDJ重组。我们还将评估RNA 修饰促进程序化DNA重排。我们结合了老鼠遗传学，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没有得到很好的控制时，会发生白血病和淋巴瘤。 拟议的研究导致更好地理解抗体基因多样化的机制， 教育我们如何预防B细胞癌（特别是在DLBCL和多发性骨髓瘤的背景下）。

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.