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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脱氨酶（AID）的表达将类别转换重组（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且可能是共同的作用。 AID 还在成纤维细胞中诱导报告构建体上的 CSR，从而证明了作为 AID 调节和下游事件目标的因子的普遍表达。基于 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，预测 AID 脱氨（编辑）胞苷，在尚未鉴定的 mRNA 上形成尿苷。编辑可以使非基因组编码的蛋白质变体激活剂表达，也可以损害抑制蛋白的表达，从而使产生企业社会责任和健康管理的关键事件。拟议的研究将确定 AID 是否具有 mRNA 编辑活性，识别在人类 B 淋巴细胞中编辑的 mRNA，并证明从编辑的 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，其中使用两个互补但不同的生物选择系统来识别由编辑产生的 mRNA 中的核苷酸多态性。实验细胞系统和激活的人类 B 淋巴细胞将用于验证所选 mRNA 上发生的编辑，并将评估新蛋白质变体在 CSR 和 SHM 中的作用。具体目标是： 1. 使用异源双链错配 DNA 选择系统分离和鉴定编辑的 mRNA，并作为补充方法，分离和表征编辑的 mRNA，这些 mRNA 是通过用 6His-tagged-AID 原位组装的亲和纯化编辑体回收的。 2. 验证候选编辑底物在实验系统和活化的人 B 细胞中被 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。这项研究的意义在于，这些发现应该为人类 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。