The Role of mRNA Editing in B Cell Development

mRNA 编辑在 B 细胞发育中的作用

基本信息

批准号：
6708008
负责人：
Harold C Smith
金额：
$ 23.63万
依托单位：
UNIVERSITY OF ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2003
资助国家：
美国
起止时间：
2003-03-01 至 2006-02-28
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：最近的研究通过激活诱导的脱氨酶（AID）的表达联系了类开关重组（CSR）和躯体超突变（SHM）。 AID在转基因AID - / - 小鼠脾脏B细胞中诱导的CSR和SHM，表明AID在这些途径中具有决定因素，也许具有共同的作用。 AID还诱导成纤维细胞中的报告基因构建体的CSR，从而证明了无处不在的因素表达，这些因素是辅助调节和下游事件的靶标。 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 CSR和SHM产生的关键事件。拟议的研究将确定AID是否具有mRNA编辑活性，确定在人B淋巴细胞中编辑的mRNA，并证明从编辑的mRNA转化的蛋白质能够介导CSR和SHM。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由编辑引起的mRNA中的核苷酸多态性。实验性细胞系统和活化的人B淋巴细胞将用于验证在选定的mRNA上进行编辑是否会进行编辑，并且将评估新型蛋白质变体在CSR和SHM中的作用。具体的目的是：1。使用异形无匹配的DNA选择系统分离和识别编辑的mRNA，作为一种互补方法，分离和表征编辑的mRNA，这些mRNA被用亲和纯化的编辑体恢复，并用6his-tagged-aid组装。 2。验证该候选者编辑底物在实验系统和激活的人B细胞中编辑，并确定在没有A1D表达的情况下编辑的mRNA诱导CSR和SHM的能力。拟议研究的意义在于，这些发现应提供对人B细胞CSR和SHM机制的重要见解，这是人类抗体反应的基本过程。此外，结果对于理解CSR和/或SHM受影响的人类免疫缺陷以及人类B细胞恶性肿瘤的发展具有重要意义，这是由这些过程的贡献引起的。