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Introduction 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. m6A has been implicated in mesenchymal transition of cells and organ fibrosis, including the kidneys, liver, and lung. Of note, m6A-targeting therapeutics are currently in advanced-stage clinical trials with good safety profile. However, the role of m6A in Systemic Sclerosis (SSc) remains unknown to date.Material and Methods We measured the expression of the main enzymes responsible for deposition of m6A (METTL3, METTL14, WTAP) and quantified global m6A levels (colorimetric assay) in peripheral blood mononuclear cells (PBMCs) derived from 31 SSc patients and 24 apparently healthy individuals (controls). We also performed transcriptome-wide mapping of the m6A methylome 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) direct RNA sequencing by Nanopore. We performed bioinformatic analysis to identify differentially methylated sites and genes, as well as the affected biological processes and 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, METTL14 and WTAP as well as global m6A 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 methylated RNA sites in SSc vs controls (absolute fold-change>1.5, P<0.05). Ingenuity pathway analysis of the differentially methylated genes identified by microarray revealed TGF-beta signaling among the top differentially methylated pathways. These results were validated by enrichment analysis of differentially methylated genes identified by Nanopore. Finally, in vitro treatment of human skin fibroblasts with STM2457 significantly ameliorated the TGF-beta-induced increase in proinflammatory-profibrotic genes, i.e. SERPINE1 and IL6, as well as markers of myofibroblast activation including ACTA2 (aSMA) and COL1A1.Conclusions SSc is characterized by increased expression of key m6A RNA methyltransferases and elevated global m6A levels, leading to widespread changes in the methylation of 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.