Direct RNA sequencing reveals multilayered epitranscriptomic remodeling in macrophages upon Mtb infection

Understanding the host transcriptional and epitranscriptomic response to <i>Mycobacterium tuberculosis</i> (Mtb) infection is vital for decoding mechanisms of immune evasion and persistence. Here, we employed Oxford Nanopore Technologies (ONT)-based direct RNA sequencing (DRS) on human THP-1 macrophages infected with Mtb. This third-generation sequencing approach enables full-length transcript analysis and simultaneous detection of RNA modifications without reverse transcription or amplification. We uncovered extensive alternative splicing events, widespread shortening of poly(A) tails, and significant shifts in usage of proximal versus distal polyadenylation sites upon infection. Furthermore, we identified infection-induced changes in m6A, m5C, pseudouridine (Ψ), and inosine modifications across different genomic regions, with distinct motif preference and distribution shifts. Pathway enrichment analyses revealed these changes were associated with host responses to infection, inflammation, metabolism, and apoptosis. Our study provides a comprehensive epitranscriptomic landscape of macrophage responses to Mtb infection and highlights potential regulatory layers governing host-pathogen interaction.