Role of ncRNA Surveillance Complex "RNA Exosome" in Class Switch Recombination and Somatic Hypermutation

ncRNA 监视复合物“RNA 外泌体”在类别转换重组和体细胞超突变中的作用

• 批准号: 9917742
• 负责人: Uttiya Basu
• 金额: $ 60.33万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-07 至 2022-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9917742
• 关键词: ATAC-seq; Address; Affinity; Affinity Chromatography; Alleles; Antibodies; Antibody Affinity; Antigens; Antisense RNA; B-Cell Activation; B-Lymphocytes; Biochemical; Catalytic Domain; Characteristics; Chromatin; Chromosomal translocation; Complex; Cytidine; DNA; DNA Damage; DNA Sequence; DNA Structure; Data; Deamination; Development; Disease; Enhancers; Event; Exons; Exonuclease; Frequencies; Genes; Genetic Transcription; Genome; Genomics; Heavy-Chain Immunoglobulins; Hyperimmunoglobulin M Syndrome; Immunoglobulin Class Switching; Immunoglobulin Genes; Immunoglobulin Somatic Hypermutation; Immunoglobulin Switch Recombination; Immunoglobulin Variable Region; Immunoglobulins; Immunologic Deficiency Syndromes; In Vitro; LacZ Genes; Lead; Light; Macromolecular Complexes; Malignant Neoplasms; Mediating; Microscopy; Molecular; Multiple Myeloma; Mus; Mutagenesis; Mutate; Mutation; Nucleosomes; Oncogenic; Pathway interactions; Patients; Point Mutation; Process; Production; Proteins; RNA; RNA Degradation; RNA Polymerase II; RNA Processing; RNA Splicing; Regulation; Research Design; Rest; Ribonucleases; Role; SETX gene; Scaffolding Protein; Single-Stranded DNA; Site; Structure of germinal center of lymph node; Techniques; Technology; Transcript; Untranslated RNA; V(D)J Recombination; Work; activation-induced cytidine deaminase; base; chromatin remodeling; cofactor; exosome; experimental study; genomic locus; helicase; large cell Diffuse non-Hodgkin' s lymphoma; melting; mouse model; prevent; promoter; protein activation; reconstitution; repaired; transcriptome; transcriptomics; tumorigenesis; variable region gene

PROJECT SUMMARY Background: Class switch recombination and somatic hypermutation are two B lymphocyte specific processes that mediate antibody diversification. Activation Induced Cytidine Deaminase (AID) is essential for initiating both of these processes by deaminating cytidine residues in immunoglobulin (Ig) loci DNA. Despite its specific and indispensible function in the Ig loci, AID has been demonstrated to also deaminate non-Ig locus genes, catalyzing various oncogenic translocations that manifest in tumorogenesis. Recent work has indicated that AID's DNA deamination activity requires its association with transcriptionally stalled RNA polymerase II and the RNA exosome complex. RNA exosome is a cellular non-coding RNA processing and/or degradation macromolecular complex. It is postulated that RNA Exosome's activity is mediated by specific cofactors and sequence characteristics of the target RNA. How RNA exosome's RNA processing activity facilitates AID mediated DNA deamination is a question we seek to address in this application. Objective/Hypothesis: In his proposal, we will determine how RNA exosome activity on transcripts generated in the IgH locus and the rest of the B cell genome generates single-stranded DNA structures following stalling of RNA polII complex and depletion of nucleosomes. Single-strand DNA structures are suitable AID substrates. Specific aims: Aim 1: Are the DIS3 and Exosc10 RNase subunits of RNA exosome complex important for AID activity?; AIM 2: How does RNA exosome substrate antisense RNA (xTSS-RNA and asRNA) promote AID targeting in the B cell genome? AIM 3: To evaluate the role of RNA exosome cofactor Mtr4 (and Senataxin) in the mutagenesis of both strands of DNA in the IgH locus and other regions of the B cell genome. Study Design: Using mouse models that are deficient in RNA exosome RNA degradation activity, we will identify regions in germinal center derived B cell genome that express RNA exosome substrate non-coding RNAs and are also mutated by AID. We will evaluate the mechanism of formation of single-strand DNA structures following localized chromatin remodeling at these identified AID target DNA sequences. We have also generated a mouse model in which the RNA exosome subunit, Exosc3, has been tandem-tagged for high affinity RNA exosome complex purification. Using B cells from these mice, we have purifed RNA exosome complex co-factors and identify them by LC-MS/MS. We will evaluate the role of RNA exosome co-factor(s) in stimulating AID/RNA exosome complex function on both strands of transcribed DNA substrate. Disease Relevance: AID initiates various malignancies in B cells due to its aberrant DNA mutagenesis activity. Proposed studies leads to a better understanding of the mechanisms initiating AID dependent oncogenesis in B lymphocytes (specially in context of DLBCL and Multiple Myeloma), as well as, have direct implications in understanding B lymphocyte based immunodeficiency syndromes like Hyper-IgM syndrome 2.

项目概要 背景：类别转换重组和体细胞超突变是两种 B 淋巴细胞特异性的 介导抗体多样化的过程。激活诱导胞苷脱氨酶 (AID) 对于 通过使免疫球蛋白 (Ig) 位点 DNA 中的胞苷残基脱氨基来启动这两个过程。尽管其 AID 在 Ig 基因座中具有特定且不可或缺的功能，已被证明还能使非 Ig 基因座脱氨基 基因，催化肿瘤发生中表现出的各种致癌易位。最近的工作表明 AID 的 DNA 脱氨基活性需要与转录停滞的 RNA 聚合酶 II 相关 和RNA外泌体复合物。 RNA 外泌体是一种细胞非编码 RNA 加工和/或降解 大分子复合物。据推测，RNA 外泌体的活性是由特定的辅因子介导的 目标RNA的序列特征。 RNA 外泌体的 RNA 加工活性如何促进 AID 介导的 DNA 脱氨基作用是我们在本申请中寻求解决的问题。 目标/假设：在他的提议中，我们将确定转录本上的 RNA 外泌体活性如何产生 IgH 基因座和 B 细胞基因组的其余部分在停滞后生成单链 DNA 结构 RNA polII 复合物的形成和核小体的消耗。单链 DNA 结构是合适的 AID 底物。 具体目标：目标 1：RNA 外泌体复合物的 DIS3 和 Exosc10 RNase 亚基对于 AID 是否重要 活动？;目标 2：RNA 外泌体底物反义 RNA（xTSS-RNA 和 asRNA）如何促进 AID 靶向 B 细胞基因组？目标 3：评估 RNA 外泌体辅因子 Mtr4（和 Senataxin）在 IgH 基因座和 B 细胞基因组其他区域中两条 DNA 链的诱变。 研究设计：使用缺乏 RNA 外泌体 RNA 降解活性的小鼠模型，我们将 鉴定生发中心衍生的 B 细胞基因组中表达 RNA 外泌体底物非编码的区域 RNA 也会因 AID 而发生突变。我们将评估单链DNA的形成机制 在这些确定的 AID 靶 DNA 序列上进行局部染色质重塑后的结构。我们有 还生成了一个小鼠模型，其中 RNA 外泌体亚基 Exosc3 已被串联标记为高 亲和RNA外泌体复合物纯化。使用这些小鼠的 B 细胞，我们纯化了 RNA 外泌体 复杂的辅因子并通过 LC-MS/MS 进行鉴定。我们将评估 RNA 外泌体辅助因子在 刺激转录 DNA 底物双链上的 AID/RNA 外泌体复合物功能。 疾病相关性：AID 由于其异常的 DNA 诱变活性而在 B 细胞中引发各种恶性肿瘤。 拟议的研究有助于更好地理解 AID 依赖性肿瘤发生的启动机制 B 淋巴细胞（特别是在 DLBCL 和多发性骨髓瘤的情况下），以及对以下方面有直接影响： 了解基于 B 淋巴细胞的免疫缺陷综合征，如高 IgM 综合征 2。