PROJECT SUMMARY Background: VDJ recombination, Class switch recombination (CSR) and somatic hypermutation (SHM) are three B lymphocyte-specific processes that mediate antibody gene diversification. VDJ recombination requires the DNA double-strand break generation by the recombination activation genes (RAG1 and RAG2) whereas CSR and SHM require the single-strand DNA break activity of the activation induced deaminase (AID) enzyme. Both RAG1/2 and AID activities are coupled with noncoding RNA transcription at sites of DNA break/mutation. The properties of the ncRNAs generated at sites of programmed DNA breaks are poorly characterized in B- cells. Recently advances in biology have provided compelling evidence that post-transcriptional and co- transcriptional regulation of chromatin associated noncoding regulatory RNAs expressed from the IgH locus (here, nomenclatured as VDJ-carRNA) and their modifications may have significant roles in driving B cell development and function and prevention of THES immunodeficiency, a severe inherited disorder. In this application, supported by preliminary data generated in our laboratory, we are evaluating the role of VDJ- carRNA in early B cell development and VDJ recombination. Objective/Hypothesis: VDJ-carRNAs expressed from the IgH locus of immature B cells are processed by the RNA exosome complex to promote VDJ recombination via mechanisms that are relevant for RAG enzyme activity. Specific aims: Aim 1: Can B cell development be rescued in exosome deficient mice; Aim 2: Are specific RNA helicases important for B cell development and VDJ recombination?; Aim 3: Do VDJ-carRNAs influence VDJ recombination efficiency? Study Design: Using cell lines and mouse models, we will study potential mechanisms by which RNA exosome and substrate chromatin associated regulatory ncRNAs (VDJ-carRNA) on the IgH locus play roles in orchestrating VDJ recombination. We will generate direct evidence that in the absence of RNA exosome and its essential helicase cofactors B cell development is attenuated due to a direct defect in VDJ recombination. We will use a combination of various genomic and biochemical tools to elucidate the associated mechanisms. Disease Relevance: B cells are a central component of the adaptive immune response, but also prone to undergo leukemias and lymphomas when antibody gene diversification processes are not well controlled. Moreover, B cells deficient in RNA exosome demonstrate trichohepatoenteric (THES) syndrome, a B cell immune system disease.