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

项目摘要 背景：类转换重组（CSR）和体细胞超突变（SHM）是两种B淋巴细胞 介导抗体多样化的特定过程。激活诱导的胞苷脱氨酶（AID）是 通过对免疫球蛋白（IG）基因座中的胞苷残基进行脱氨基，对启动这两个过程至关重要 DNA.尽管AID在IG基因座中具有特异性和不可或缺的功能，但已证明AID也 脱氨基非Ig基因座基因，伴随着催化各种致癌基因的阴性结果， 在肿瘤发生中表现出来的易位。最近的研究表明AID的DNA脱氨活性 需要其与转录停滞的RNA聚合酶II和RNA外泌体复合物的结合。RNA 外泌体是细胞非编码RNA（ncRNA）加工和/或降解大分子复合物。如何 ncRNA和RNA外泌体促进AID的DNA脱氨活性是我们计划解决的问题。 目的/假设：我们将确定ncRNA上的RNA外泌体活性如何在细胞内支持AID活性。 IgH基因座，并稳定B细胞基因组的三维结构。具体目标：目标1：了解 非编码RNA和RNA外泌体在控制AID活性中的染色质相关功能。目标2： 了解CTCF结合元件（cbe）ncRNA在TADIgH组织中的作用。目标3：了解 新的RNA外泌体辅因子在调节AID活性中的作用。 研究设计：使用RNA外泌体介导的RNA降解缺陷的小鼠模型， 鉴定了B细胞基因组中表达外泌体敏感性ncRNA并且也被AID突变的区域。 我们将评估单链DNA结构的形成机制， 在这些鉴定的AID靶DNA序列处重塑。我们发现了一个新的外来体家族 与CTCF结合元件（CBE）相关的敏感ncRNA，定义为cbeRNA。我们将 评估cbeRNA加工如何调节IgH的三维拓扑相关结构域结构 并影响SHM和CSR效率。最后，使用来自TAP标记的外泌体表达小鼠的B细胞， 模型，我们已经纯化和鉴定了染色质相关的RNA外泌体辅因子。我们将评估 RNA外泌体辅因子在刺激AID/RNA外泌体复合物功能中的作用。 疾病相关性：拟议的研究将导致更好地了解启动机制 B淋巴细胞中的AID依赖性肿瘤发生（特别是在DLBCL和多发性骨髓瘤的情况下）， 并对理解基于B淋巴细胞的免疫缺陷综合征， 高IgM综合征2型。RNA外泌体与许多疾病有关，包括许多 神经退行性疾病和多发性骨髓瘤以及该资助的发现将带来更好的治疗 了解疾病的病因和治疗方法的发展。

PROJECT SUMMARY Background: Class switch recombination (CSR) and somatic hypermutation (SHM) 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, with the concomitant negative outcome of catalyzing various oncogenic translocations that manifest in tumorigenesis. 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 (ncRNA) processing and/or degradation macromolecular complex. How ncRNA and RNA exosome facilitate AID's DNA deamination activity is a question we plan to address. Objective/Hypothesis: We will determine how RNA exosome activity on ncRNAs support AID activity at the IgH locus and stabilizes 3-dimensional organization of the B cell genome. Specific aims: AIM 1: To understand the chromatin associated function of noncoding RNAs and RNA exosome in controlling AID activity. AIM 2: To understand the role of CTCF binding element (cbe) ncRNAs in TADIgH organization. AIM 3: To understand the role of novel RNA exosome cofactors in regulating AID activity. Study Design: Using mouse models that are deficient in RNA exosome-mediated RNA degradation, we have identified regions in the B cell genome that express exosome sensitive ncRNAs and also are 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 discovered a new family of exosome sensitive ncRNAs that are associated with CTCF-binding elements (CBE), defined as cbeRNAs. We will evaluate how cbeRNA processing regulates the 3 dimensional topologically associating domain structure of IgH and influences SHM and CSR efficiencies. Finally, using B cells from TAP-tagged Exosome expressing mouse model, we have purified and identified chromatin associated RNA exosome co-factors. We will evaluate the role of RNA exosome co-factor(s) in stimulating AID/RNA exosome complex function. Disease Relevance: The proposed studies will lead to a better understanding of the mechanisms initiating AID dependent oncogenesis in B lymphocytes (particularly in the contexts of DLBCL and multiple myeloma) as well as have direct implications in understanding B lymphocyte-based immunodeficiency syndromes like Hyper-IgM syndrome type 2. RNA exosome is implicated in many diseases including numerous neurodegenerative disorders and multiple myeloma and findings from this grant will lead to a better understanding of the etiologies of the disorders and development of therapies.