RNA m ⁶ A and 5hmC regulate monocyte and macrophage gene expression programs

Background RNA modifications are essential for the establishment of cellular identity. Although increasing evidence indicates that RNA modifications regulate the innate immune response, their role in monocyte-to-macrophage differentiation and polarisation is unclear. To date, most studies have focused on m 6 A, while other RNA modifications, including 5hmC, remain poorly characterised. The interplay between different RNA modifications that may occur in specific cellular contexts remains similarly unexplored. Results We profiled m 6 A and 5hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. We observed that decreased expression of m 6 A and 5hmC writers, METTL3 and TET-enzymes respectively, facilitated monocyte-to-macrophage differentiation. Despite a global trend of m 6 A and 5hmC loss during macrophage differentiation, enrichment of m 6 A and/or 5hmC on specific categories of transcripts essential for macrophage differentiation positively correlated with their expression and translation. m 6 A and 5hmC mark and are associated with the expression of transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discovered the coexistence of m 6 A and 5hmC marking alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of transcripts with key roles in macrophage biology. In specific examples, RNA 5hmC controls the decay of transcripts independently of m 6 A. Conclusions This study: i) uncovers m 6 A, 5hmC and their writer enzymes as regulators of monocyte and macrophage gene expression programs and ii) provides a comprehensive dataset to interrogate the role of RNA modifications in a plastic system. Altogether, this work sheds light on the role of RNA modifications as central regulators of effector cells in innate immunity.