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

项目总结 背景：VDJ重组、类切换重组(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 重组激活基因(RAG1和RAG2)产生DNA双链，其中作为CSR和 SHM需要激活诱导脱氨酶(AID)酶的单链DNA断裂活性。两者都有 在DNA断裂/突变部位，RAG1/2和AID活性与非编码RNA转录结合。这个 在B细胞中，在程序性DNA断裂部位产生的ncRNAs的性质特征很差。 最近生物学的进步提供了令人信服的证据，证明转录后和共转录 NcRNAs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，由我们的 实验室，我们正在评估RNA修饰N6-甲基腺苷(M6A)及其相关基因的作用 酶METTL3和METTL14在B细胞发育、功能和基因组完整性中的作用。 目标/假设：在他的建议中，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基因控制着程序性DNA重组， 抗体基因多样化，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对班级切换重要吗 重组，体细胞超突变，和/或防止B细胞基因组稳定？目的2：什么是 M6A修饰促进IgH DNA重组和/或阻止基因组的机制 不稳定？目的3：RNA甲基化和偶联RNA监视通路在早期B细胞中起作用吗 发展和VDJ重组期间？ 研究设计：利用细胞系和小鼠模型，研究RNA m6A甲基化的机制 在程序性DNA重组和保护B细胞基因组完整性方面发挥作用。我们将评估 艾滋病病区单链DNA结构形成机制及RNA监测 成熟B细胞的活性及其抑制RNA在RAG活性部位的降解机制 在VDJ重组过程中的B细胞。我们还将评估RNA的分子机制 修饰促进了程序性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细胞是获得性免疫反应的中心组成部分，但也容易 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。 建议的研究有助于更好地理解抗体基因多样化的机制，同时也 告诉我们如何预防B细胞癌(特别是在DLBCL和多发性骨髓瘤的情况下)。

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.