N6-methyladenosine (m6A) RNA modification is required to final plasma cell differentiation Free

Plasma cells are highly specialized cells representing the end stage of B cell differentiation. They play an important role in humoral immunity by synthesizing and secreting antibodies protecting the host against infections. On the transcriptional level, the differentiation of B cells into plasma cells (PC) is associated with substantial and coordinated changes in the gene expression profile, which fall into two main categories: the loss of B cell-associated transcripts and the acquisition of plasma cell gene expression program. Many recent studies have demonstrated that RNA undergoes various modifications in a manner similar to DNA. These RNA modifications play a role in many cellular and biological processes, thereby opening up an emerging research field known as epitranscriptomics.

We have shown that PC generation can be modeled using multi-step culture systems where various combinations ofactivation molecules and cytokines are subsequently applied in order to reproduce the sequential cell differentiation occurring in the different organs/tissues invivo. In these culture models, memory B cells (MBCs) differentiate intoCD20^low/-CD38^- pre-plasmablasts (prePBs), CD20^-CD38⁺CD138^- plasmablasts (PBs), CD20^-CD38⁺CD138⁺ early PCsand, finally, into long-lived PCs (LLPCs), which may survive and produce continuously high amounts of immunoglobulins(Igs) for months invitro. Using this model coupled with single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq), we have provided significant insights into the trajectories of PC differentiation. Our findings, published by Alaterre et al. in Blood (2024), elucidated the epigenetic and transcriptional reprogramming events underpinning PC differentiation under physiological conditions. Thanks to these data we also found significant expression changes of enzymes involved in RNA editing especially enzymes implicated in N⁶-methyladenosine (m6A) management during the PC differentiation. This is the most common RNA modification that can affect different molecular and cellular process. A few recent studies describe the importance of m6A RNA modification for Early B cell differentiation but investigations during the latest stage of human PC differentiation remain poor.

We performed RNA Mass-spectrometry analysis from each cell populations and identified that the total level of m6A mark increase during B to PC differentiation. m6A-Methylated RNA immunoprecipitation sequencing (MeRIPseq) allowed the analysis of all the transcripts that are modified by m6A mark, as well as m6A position together with the percentage of transcript modified for a given transcript. We found more than 75 000 m6A-mRNA-associated modifications and 20 000 for LncRNA with a majority specifically enriched in the preplasmablastic stage. Among m6A-mRNA that are differentially modified during B to PC differentiation we found transcript directly involved in PC differentiation and biological functions such as JCHAIN, IRF4, PRDM1, BTF3, IRF4, AICDA, but also some transcripts involved in metabolism (CD38, ARG2, FASTKD1, AK4), and DNA recombination and repair highlighting the importance of m6A mark management during B to PC differentiation.

In order to investigate the role of m6A modification during PC differentiation, we used a specific inhibitor of the m6A methyltransferase METTL3, that is the main m6A-RNA transferase. We found that the inhibition of m6A deposit leads to a significant impediment of PC differentiation. METTL3 inhibitor affects plasma cell differentiation through inhibition of maturation after the preplasmablastic stage. At the cellular level, the percentage of preplasmablasts at day 7 is significantly higher compared to the control suggesting an inhibition of the maturation in plasmablasts and PCs. These results correlates with the fact that the preplasmablasts express the highest level of the enzymes involved in m6A management, such as METTL3 but also the demethylase FTO (Fat mass and Obesity-associated) and several specific readers like YTHDC1, underlining the importance of m6A-RNA modifications in the preplasmablastic stage.

Single-cell RNAseq analysis of METTL3i-treated cells coupled with proteomic analysis are currently ongoing to decipher the molecular and transcriptional circuits associated with this maturation block mediated by m6A inhibition. Overall this comprehensive analysis will enhance our understanding of the role of m6A during B to PC differentiation.