Title: The role of RNA base modification m⁶A in RNA turnover and genome dynamics in B cell programmed DNA recombination
Description: Transcription-associated RNA surveillance is vital to the well-being of the cell by processing both coding and noncoding RNA (ncRNA). When left unregulated, RNA transcripts can cause genomic instability by forming structures called R-loops that leave a single strand of DNA exposed to potentially harmful events like nicks and mutations. The RNA exosome is the 3’ exoribonuclease that plays an important role in degrading both coding and ncRNA, and its role in processing ncRNA transcripts has been demonstrated extensively in the recent past. The role of ncRNA transcription and the RNA exosome is best exemplified in B cell programmed DNA recombination, namely class switch recombination (CSR). In CSR, ncRNA transcription and RNA exosome recruitment to switch sequences allows for targeting of the activation-induced cytidine deaminase (AID) enzyme and ultimate formation of DNA double strand breaks, which results in recombination between the appropriate switch regions. While the mechanism of the RNA exosome in processing this transcribed ncRNA and regulating CSR is well-established, the mechanism of RNA exosome regulation and recruitment remains unknown.

RNA base modifications are another facet of RNA surveillance that can have profound effects on RNA biology. While some modifications are fixed, reversible base modifications such as methylation are particularly influential due to their dynamic nature. Among the most widely studied of reversible modifications is that of N⁶-methyladenosine (m⁶A). M⁶A has been identified for its role in regulation of gene expression and cell differentiation via its effect on regulating mRNA levels and downstream function. Although it has been shown to be present on some ncRNA, the role of m6A in regulating ncRNA turnover and genome dynamics is largely unclear. We interrogated the hypothesis that m6A plays a role in regulating RNA exosome recruitment in B cell programmed DNA recombination. We initially assessed the role of the RNA exosome cofactor MPP6 in CSR using CRISPR/Cas9-mediated knockout mouse B cell hybridoma CH12F3 cells (CH12 cells) and established a connection between the RNA exosome and m6A by demonstrating an interaction between MPP6 and m6A reader protein YTHDC1.

We then utilized mouse models to knockout METTL3, a m6A methyltransferase, in primary B cells and assessed the efficiency of CSR. We performed RNA sequencing to assess the levels of ncRNA in B cells upon METTL3 loss and find that primarily G-rich RNA transcripts are accumulated upon METTL3 loss, exemplified by switch region transcript SμGLT. In order to specifically show that methylation of SμGLT is required for efficient CSR, we used fusion proteins of catalytically dead Cas13b fused to either m6A demethylase ALKBH5 or FTO and targeted SμGLT with a guide RNA specific for the transcript. We found that methylation of SμGLT specifically is required for efficient CSR, with targeted demethylation resulting in a decrease in CSR efficiency. We also interrogated the role of m6A reader protein YTHDC1 in CSR via CRISPR/Cas9-mediated knockout CH12 cells to assess the mechanism of m6A-mediated downstream function. We found that YTHDC1 is also required for efficient CSR, likely via interaction with MPP6 and the RNA exosome to degrade SμGLT.

We also assessed the role of MPP6, METTL3, and YTHDC1 in facilitating the association between AID and the RNA exosome via 3D stochastic optical reconstruction microscopy (3D-STORM) and found that all three components are required for nucleation of AID and the RNA exosome (for which helicase MTR4 was used as a proxy). We then wanted to assess whether m⁶A loss promotes genomic instability in B cell programmed DNA breaks. We performed linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) to assess the levels of on- versus off-target rearrangements in METTL3 knockout primary B cells. We found that upon METTL3 loss there is an increase in off-target, non-immunoglobulin translocations as well as an increase in microhomology-mediated end joining, indicative of defects in on-target recombination and the canonical nonhomologous end joining pathway. These data suggest that m⁶A is required for targeted programmed DNA rearrangements and protection of nonhomologous end joining in B cells.

Taken together, these findings have elucidated a novel mechanism by which m6A regulates ncRNA turnover, RNA exosome targeting, and genome dynamics in B cell programmed DNA rearrangements.