It is established that Leishmania infection drives extensive yet selective remodeling of the host m6A landscape and position the host epitranscriptome as a previously unrecognized regulatory axis in Leishmania-macrophage interactions, with implications for the development of host-directed therapeutic strategies.
Abstract
N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA, yet its role in host responses to protozoan infection remains poorly explored. Here, we provide the first characterization of the host m6A epitranscriptome during Leishmania amazonensis infection, integrating clinical data, in vitro macrophage models, and direct RNA sequencing. Analysis of publicly available transcriptomic datasets from patients with cutaneous and visceral leishmaniasis, validated in an independent cohort of patients with cutaneous leishmaniasis, revealed coordinated remodeling of the m6A regulatory machinery, characterized by significant upregulation of the writer METTL3 and downregulation of the eraser ALKBH5 and the reader YTHDF3. These changes were recapitulated in RAW264.7 macrophages infected in vitro, where METTL3 protein levels increased progressively between 2 and 8 hours post-infection (hpi), while ALKBH5 abundance declined, resulting in elevated global m6A levels detectable as early as the first post-internalization time point. Transcriptome-wide mapping by direct RNA sequencing at 8 hpi identified 1,998 m6A-modified transcripts harboring 7,132 high-confidence sites in non-infected macrophages, compared with 2,956 modified transcripts and 13,495 sites in infected cells. Beyond this quantitative expansion, infection was associated with increased modification occupancy at shared sites, a greater prevalence of multi-site and multi-region methylation, and selective remodeling of immune-related and immunometabolic transcripts, including Tlr4, Ccl2, Hmox1, Socs3, Rela, Hk1, Hk2, and Dicer1. Altogether, these findings establish that Leishmania infection drives extensive yet selective remodeling of the host m6A landscape and position the host epitranscriptome as a previously unrecognized regulatory axis in Leishmania-macrophage interactions, with implications for the development of host-directed therapeutic strategies. Impact Statement N6-methyladenosine (m6A) is the most abundant modification in eukaryotic mRNA, and emerging evidence links epitranscriptomic remodeling to host responses against diverse pathogens. However, whether m6A regulates host-macrophage responses during Leishmania infection remains unknown. Using Oxford Nanopore Direct RNA sequencing, we provided the first transcriptome-wide map of host m6A modifications during Leishmania amazonensis infection. Infection selectively remodels the m6A landscape of macrophages, targeting immune-regulatory and immunometabolic transcripts, establishing the host epistranscriptome as a previously unrecognized regulatory layer in Leishmania-macrophage interactions.
BACKGROUND
The global emergence of monkeypox virus (MPXV) highlights the urgent need for a deeper understanding of host-pathogen interactions. Although transcriptional responses to MPXV infection have been characterized, the role of epitranscriptomic regulation particularly N6‑methyladenosine (m6A) modification remains largely unexplored.
METHODS
We performed an integrated analysis of time‑series transcriptomic and m6A methylome profiles using whole blood samples collected from MPXV‑infected rhesus macaques at 7, 14, and 21 days post‑infection, with distinct animals used at each time point. Host gene expression and m6A modification dynamics were examined over the course of infection. Differential expression and differential m6A modification analyses were conducted, followed by integrative pathway and immune cell signature profiling.
RESULTS
MPXV infection induced sustained host reprogramming, characterized by suppression of immune pathways and activation of metabolic processes. A global increase in m6A modifications was observed, accompanied by upregulation of the methyltransferase METTL3 and downregulation of demethylases (FTO, ALKBH5) and readers (YTHDF1-3). Knockdown of METTL3 or YTHDF2 reduced viral replication, suggesting a proviral role for this regulatory circuit. Integrative analysis identified 38 genes with coordinated changes in both transcription and m6A modification across all three time points. Focusing on literature-curated pathogenic pathways, we further identified 11 dual-regulated host factors. Notably, DNAJB1 was the only gene shared between these two independent selection strategies. m6A peaks near transcription start sites and within 5'UTR positively correlated with gene expression, whereas coding region modifications showed weak negative correlations. Immune lineage signatures showed gradual declines in T cell, NK, and monocyte/macrophage signatures with a progressive increase in B cell signatures. Cross‑dataset comparison confirmed core m6A regulatory trends despite heterogeneity across tissues and viral strains.
CONCLUSIONS
This study reveals m6A epitranscriptomic remodeling as a key correlate of the host response to MPXV infection and nominates DNAJB1 alongside the other 10 dual‑regulated genes as candidate host factors for further mechanistic investigation.
R. Tang, Jin-Di Huang, Wenhai Yu et al.· Cell communication and signa...· 0 citations
GLORI sequencing is applied to generate single-base resolution transcriptome-wide m6A maps in seven bacterial species to provide a quantitative atlas of bacterial m6A and establish a foundation for understanding its regulatory and evolutionary roles.
Youyue Li, Letong Xu, Na Liu et al.· Cell Reports· 0 citations
N6-methyladenosine (m
6
A), the most abundant internal RNA modification in eukaryotic cells, has emerged as a critical regulator of antiviral host defense. Increasing evidence indicates that m
6
A functions beyond conventional post-transcriptional regulation by dynamically coordinating the magnitude, timing, and duration of immune responses during viral infection. In SARS-CoV-2 infection, dysregulated antiviral immunity is characterized by delayed interferon activation together with sustained inflammatory responses, suggesting the existence of regulatory mechanisms that continuously calibrate immune signaling rather than simply switching it on or off. In this review, we propose the concept of m6A as a potential “epitranscriptomic immune rheostat,” representing a conceptual framework in which m6A may fine-tune antiviral immunity through coordinated regulation of RNA stability, translational efficiency, and transcript turnover. We summarize how m
6
A shapes multiple layers of innate immune responses, including pattern recognition receptor sensing, type I interferon signaling, inflammatory buffering, and immune resolution. We further discuss emerging evidence suggesting that SARS-CoV-2 infection is associated with dynamic modulation of the m
6
A regulatory machinery and viral RNA methylation landscapes, while emphasizing that many mechanistic insights remain to be experimentally validated in SARS-CoV-2 models. In addition, we highlight the interplay between m
6
A regulation and immunometabolic remodeling, while distinguishing direct SARS-CoV-2 evidence from findings derived from other viral systems or broader m
6
A biology. Selected comparisons with Influenza A virus are discussed as complementary evidence to explore potentially conserved principles of m
6
A-mediated immune regulation among RNA viruses rather than as direct evidence for SARS-CoV-2 infection. Finally, we discuss the therapeutic implications of targeting the m
6
A regulatory network to recalibrate immune responses with temporal and cellular precision, while noting that current m
6
A-targeting strategies remain at the preclinical proof-of-concept stage and require further evaluation regarding specificity, safety, and translational feasibility. Collectively, this review provides a hypothesis-driven conceptual framework for understanding how m
6
A integrates RNA fate control with antiviral immunity and immunopathology during SARS-CoV-2 infection.
Unknown authors· Frontiers in Immunology· 0 citations
It is argued that while epitranscriptomics represents a compelling regulatory axis in cancer cell death, advancing the field will require integrative, high-resolution, and functionally precise approaches to move beyond correlative frameworks toward mechanistic and clinically actionable insights.
A. Mukherjee, Ankit Kumar Bharti, D. Mathew et al.· Functional & Integrative Gen...· 0 citations
Summary Knowledge on the cell types and cell-specific gene expression of multicellular pathogens facilitates drug discovery and allows gaining a deeper understanding of pathogen biology. By utilizing single-cell RNA sequencing (scRNA-seq), we analyzed 19,581 cells of a globally prevalent parasitic flatworm, the liver fluke Fasciola hepatica, which affects health of both humans and animals. We identified 15 distinct clusters, including stem cells, gonadal, muscle and intestinal cells. Differentiation lineages within this parasite were identified and characterized by integrating RNA velocity and spatial transcriptomics data. Furthermore, an ELF5- and TRPMPZQ-expressing cell cluster was discovered, characterized by high expression of protein kinases, including the p21-activated kinase PAK4. Treatment with a PAK4 inhibitor efficiently killed the parasites. These data provide insight into the cellular composition of a complex multicellular pathogen and demonstrate how gene expression at single-cell resolution can serve as a resource for the identification of new drug targets.
Oliver Puckelwaldt, Svenja Gramberg, Sagar Ajmera et al.· iScience· 0 citations
Post-transcriptional RNA modifications modulate diverse aspects of RNA metabolism. N6-methyladenosine (m6A), one of the most abundant internal RNA modifications, is deposited by the core methyltransferase complex, METTL3 and METTL14. Oxford Nanopore Technologies (ONT) platform permits direct, single RNA molecule sequencing while preserving native modifications. However, without rigorous benchmarking, the accuracy and reproducibility of modification detection remain uncertain. Here, we leveraged ONT to comprehensively profile bona fide m6A modifications in cellular RNAs at single-nucleotide resolution by integrating two direct RNA sequencing chemistries (RNA002 and RNA004) with the m6Anet and Dorado modification-detection models. We independently depleted METTL3 and METTL14 in human cells and rigorously validated modification calls through several assays and independent orthogonal methods (GLORI and miCLIP). We find that Dorado detected a higher number of m6A events and enabled simultaneous detection of other RNA modifications (5-methylcytosine, pseudouridine, and inosine). Pairing Dorado with an in vitro transcribed, unmodified control under stringent filtering, we provide compelling evidence supporting a global reduction in m6A sites and stoichiometry within coding sequences and across genes, particularly in highly modified genes and sites, and at consensus DRACH motifs. We report a differential and complex regulation of modified transcripts, accompanied by a global reduction in poly(A) tail length. Notably, METTL3 and METTL14 depletion produced distinct transcript-specific effects, supporting non-redundant roles within the m6A writer complex. Together, our study illustrates a notable advancement of ONT capabilities and establishes a robust transcriptome-wide framework for RNA modification detection, thereby laying the groundwork for exploring the contribution of METTL3/METTL14 to cellular functions and disease.
Emily Kurtyan, Andrew J. Stein, Kelly J. Abdalla et al.· PLoS Genetics· 0 citations
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