Role of mRNA Editing in B Cell Development

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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.