Dynamic changes in RNA mA and 5 hmC influence gene expression programs during macrophage differentiation and polarisation.

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. While mA has been widely studied, other RNA modifications, including 5 hmC, remain poorly characterised. We profiled mA and 5 hmC epitranscriptomes, transcriptomes, translatomes and proteomes of monocytes and macrophages at rest and pro- and anti-inflammatory states. Transcriptome-wide mapping of mA and 5 hmC reveals enrichment of mA and/or 5 hmC on specific categories of transcripts essential for macrophage differentiation. Our analyses indicate that mA and 5 hmC modifications are present in transcripts with critical functions in pro- and anti-inflammatory macrophages. Notably, we also discover the co-occurrence of mA and 5 hmC on alternatively-spliced isoforms and/or opposing ends of the untranslated regions (UTR) of mRNAs with key roles in macrophage biology. In specific examples, RNA 5 hmC controls the decay of transcripts independently of mA. This study provides (i) a comprehensive dataset to interrogate the role of RNA modifications in a plastic system (ii) a resource for exploring different layers of gene expression regulation in the context of human monocyte-to-macrophage differentiation and polarisation, (iii) new insights into RNA modifications as central regulators of effector cells in innate immunity.