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

## TL;DR

This study uses advanced RNA sequencing to uncover how tuberculosis infection alters gene regulation in immune cells.

## Contribution

The novel use of direct RNA sequencing reveals epitranscriptomic changes in macrophages during Mtb infection.

## Key findings

- Mtb infection causes extensive alternative splicing and poly(A) tail shortening in macrophages.
- RNA modifications like m6A and m5C shift significantly during infection.
- Epitranscriptomic changes are linked to immune response, inflammation, and cell death pathways.

## Abstract

Understanding the host transcriptional and epitranscriptomic response to Mycobacterium tuberculosis (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.