The Role of mRNA Editing in B Cell Development

描述（由申请人提供）：最近的研究通过激活诱导脱氨酶（AID）的表达将类开关重组（CSR）和体细胞超突变（SHM）联系起来。AID在转基因AID-/-小鼠脾B细胞中诱导CSR和SHM，表明AID在这些途径中具有决定性作用，并且可能具有共同作用。AID还在成纤维细胞中诱导了报告基因构建的CSR，从而证明了作为AID调控和下游事件目标的因子的普遍表达。基于AID与apobec1（参与apoB mRNA编辑的催化亚基）的同源性和AID的胞苷脱氨酶活性，预测AID在尚未鉴定的mRNA上脱氨（编辑）胞苷以形成尿苷。编辑可能使非经济编码的蛋白质变体激活子表达或损害抑制蛋白的表达，从而使CSR和SHM产生的关键事件成为可能。该研究将确定AID是否具有mRNA编辑活性，鉴定在人B淋巴细胞中被编辑的mRNA，并证明由编辑mRNA翻译的蛋白质介导CSR和SHM的能力。提出了一种创新的实验设计，其中两个互补但不同的生物选择系统用于鉴定编辑产生的mRNA中的核苷酸多态性。实验细胞系统和活化的人类B淋巴细胞将用于验证在所选mrna上发生的编辑，并将评估新蛋白变体在CSR和SHM中的作用。具体目标是：1。使用异双工错配DNA选择系统分离和鉴定编辑过的mRNA，并作为一种补充方法，分离和鉴定编辑过的mRNA，这些编辑体是用6his标记的aid原位组装的亲和纯化编辑体恢复的。2. 验证候选编辑底物在实验系统和活化的人B细胞中可以被AID编辑，并确定编辑的mrna在没有A1D表达的情况下诱导CSR和SHM的能力。这项研究的重要意义在于，这些发现将为人类B细胞CSR和SHM的机制提供重要的见解，这是人类抗体反应的重要过程。此外，这些结果对于理解CSR和/或SHM受到影响的人类免疫缺陷以及这些过程中产生的人类b细胞恶性肿瘤的发展具有重要意义。

DESCRIPTION (provided by the applicant): Recent studies have linked class switch recombination (CSR) and somatic hypermutation (SHM) through the expression of Activation Induced Deaminase (AID). AID induced CSR and SHM in transgenic AID-/- mouse splenic B cells, suggesting that AID has a determinant and perhaps common role in these pathways. AID also induced CSR on reporter constructs in fibroblasts, thereby demonstrating the ubiquitous expression of factors that are targets for AID regulation and downstream events. Based on AID's homology to APOBEC-1 (the catalytic subunit involved in apoB mRNA editing) and AID's cytidine deaminase activity, it has been predicted that AID deaminates (edits) cytidine to form uridine on a yet-to-be identified mRNA Editing could either enabling the expression of a non-gcnomically encoded protein variant activator or impair the expression of a suppressor protein and thereby enable critical events in the generation of CSR and SHM. The proposed research will determine whether AID has mRNA editing activity, identify the mRNAs that are edited in human B lymphocytes, and demonstrate the ability of proteins translated from edited mRNAs to mediate CSR and SHM. An innovative experimental design is proposed wherein two complementary but distinct biological selection systems are used to identify nucleotide polymorphisms in mRNA arising from editing. Experimental cell systems and activated human B lymphocytes will be used to validate that editing occurs on the selected mRNAs and the role of the novel protein variants in CSR and SHM will be assessed. The Specific Aims are to: 1. Isolate and identify edited mRNAs using a heteroduplex mismatch DNA selection system and as a complementary approach, isolate and characterize edited mRNA(s) that are recovered with affinity purified editosomes assembled in situ with 6His-tagged-AID. 2. Validate that candidate editing substrates arc edited by AID in experimental systems and in activated human B cells and determine the ability of edited mRNAs to induce CSR and SHM in the absence of A1D expression. The significance of the proposed research is that the findings should provide critical insights into the mechanisms of human B cell CSR and SHM, which are essential processes m human antibody responses. In addition, the results have significance for understanding human immunodeficiency in which CSR and/or SHM are affected and the development of human B-cell malignancies that arise from a contribution from these processes.