Background: RNA methylation at the N-6 position of adenosine (m6A) is the most abundant modification in human mRNA affecting multiple aspects of RNA metabolism such as transcript stability, splicing and translation efficiency. m6A affects critical cellular functions including cell cycle progression, metabolism, and immune responses. Although deregulation of m6A has been previously reported in various human diseases and enzymatic inhibitors of the main m6A methyltransferase, METTL3, are currently under trial in advanced stages of cancer, the role of m6A in systemic autoimmune diseases remains largely unknown.

Objectives: To study the regulatory role of m6A RNA methylation in systemic sclerosis (SSc).

Methods: We measured the expression of m6A ‘writers’ (METTL3, METTL14) and quantified global m6A levels (colorimetric assay) in peripheral blood mononuclear cells (PBMCs) derived from 31 SSc patients and 24 controls. We then performed site-specific m6A analysis at single-nucleotide resolution utilizing both a) a microarray-based quantification method that identifies more than 11,000 unique m6A sites mapping on >4,500 unique genes and b) nascent RNA sequencing using Nanopore technology. We performed bioinformatic analysis to identify differentially methylated sites and transcripts, as well as the most affected pathways. Finally, we treated primary human dermal fibroblasts (HDFa) with TGF-b1 and STM2457, an enzymatic inhibitor of METTL3, to examine the effect of m6A RNA methylation on TGF-b-induced gene expression.

Results: Expression of the m6A writers METTL3 and METTL14 as well as global m6A RNA methylation levels were increased by 1.3-2.3-fold in SSc vs controls (all P<0.001). Transcriptome-wide single-nucleotide m6A analysis revealed several hundred differentially RNA methylated sites in SSc vs controls (absolute fold-change>1.5, P<0.05). Enrichment analysis of the differentially methylated transcripts revealed TGF-beta signaling among the top differentially methylated pathways. Further analysis of Nanopore data validated changes in the methylation of key TGF-b pathway components, such as TGFB1 and TGFBR2 , in SSc vs control PBMCs. Finally, in vitro treatment of human dermal fibroblasts with the enzymatic METTL3 inhibitor STM2457 significantly ameliorated the TGF-beta-induced increase in profibrotic genes including ACTA2 (αSMA) and COL1A1 .

Conclusion: These findings demonstrate that SSc is characterized by increased expression of key m6A RNA methyltransferases and elevated global m6A levels, leading to widespread changes in transcripts involved in TGF-b signaling. Notably, pharmacologic inhibition of METTL3 mitigates TGF-beta–driven profibrotic gene expression in dermal fibroblasts, suggesting that m6A-modulating strategies may hold therapeutic potential in SSc.

REFERENCES: NIL.

Acknowledgements: KSte has been supported by grants from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (MODVASC, grant agreement No 759248), the German Research Foundation DFG (CRC1366 C07, project number 394046768), the Health+Life Science Alliance Heidelberg Mannheim GmbH and the Helmholtz Institute for Tranalational AngioCardioScience (HI-TAC). PPS is supported by educational grants (Special Account for Research Grants - National and Kapodistrian University of Athens, Greece; Nr 0974).

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.