The role of N6-methyladenosine RNA modification in programmed and aberrant DNA mutagenesis in B cells

Columbia University Health Sciences, New York NY

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Abstract

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 generation by the Recombination activation genes (RAG1 and RAG2) whereas CSR and SHM requires 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 modification of ncRNAs determine a component of RNA epigenomics and have significant role in driving cellular development and function. In this application, supported by published study (Nair et al,2021; PMID: preliminary data generated in our laboratory, we are evaluating the role of RNA modification N6-methyladenosine (m6A) and its associated enzymes METTL3 and METTL14 in B cell development, function and genomic integrity. Objective/Hypothesis: In his proposal, we will determine how RNA methylation m6A on transcripts generated in the IgH locus and the rest of the B cell genome controls programmed DNA recombination, antibody gene diversification and prevents chromosomal instability. Specific aims: Aim 1: Does m6A reader protein YTHDC1 drive CSR and SHM? Aim 2: What is the mechanism of function of m6A modification and YTHDC1 in promoting IgH diversity and preventing genomic instability in B cells? Study Design: Using cell lines and mouse models, we will establish that along with m6A writers (shown previously), the m6A reader YTHDC1 protein is important for class switch recombination and somatic hypermutation. Thereafter, using a combination of genome and RNA editing toolbox generated with CRISPR/Cas9 technology, we will evaluate the effect of RNA methylation and YTHDC1 protein to induce CSR. A RNA recognition motif of YTHDC, which is necessary for efficient CSR, will be evaluated for properties that drive CSR. Whether YTHDC1 promotes RNA dependent granule formation for promoting CSR (or SHM) will be evaluated using STORM microscopy. A cell-based assay system to evaluate SHM has been developed, the role of YTHDC1 and other m6A related proteins will be evaluated for SHM efficiency. 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. Proposed studies leads to a better understanding of the mechanism of antibody gene diversification but also educate us how B cell cancer (specially in context of DLBCL and Multiple Myeloma) are prevented.