The Role of mRNA Editing in B Cell Development

描述(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)联系在一起。AID诱导了转基因AID-/-小鼠脾B细胞的CSR和SHM，提示AID在这些途径中具有决定因素，可能是共同的作用。AID还在成纤维细胞中诱导了报告结构的CSR，从而证明了作为AID调控靶点的因子和下游事件的普遍表达。根据AID与APOBEC-1(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，已预测AID将胞苷脱氨基(编辑)在尚未鉴定的mRNA上形成尿苷编辑既可以使非基因编码的蛋白变体激活物表达，也可以抑制抑制蛋白的表达，从而使CSR和SHM的生成过程中的关键事件发生。这项拟议的研究将确定AID是否具有信使核糖核酸编辑活性，鉴定人B淋巴细胞中被编辑的信使核糖核酸，并证明从编辑的信使核糖核酸翻译的蛋白质介导CSR和SHM的能力。提出了一种创新的实验设计，其中使用了两个互补但不同的生物选择系统来鉴定来自编辑的mRNA的核苷酸多态。实验细胞系统和激活的人类B淋巴细胞将被用来验证在选定的mRNAs上发生编辑，并将评估新的蛋白质变体在CSR和SHM中的作用。1.利用异源双链错配选择系统分离和鉴定编辑过的mRNAs，作为补充，分离和鉴定用亲和纯化的编辑小体与6His-tag-AID原位组装的编辑过的mRNAs(S)，并对其进行鉴定。2.在实验系统和激活的人B细胞中验证候选编辑底物是由AID编辑的，并确定在没有A1D表达的情况下编辑的mRNAs诱导CSR和SHM的能力。这项拟议的研究的意义在于，这些发现将为人类B细胞CSR和SHM的机制提供关键的见解，这两个机制是人类抗体反应的关键过程。此外，这些结果对于了解CSR和/或SHM受到影响的人类免疫缺陷以及由这些过程引起的人类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.