Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

Zhang, Wen-Di; Zhang, Dan-Dan; Wang, Xu; Li, Chen-Yang; Li, Fan-Qin; Liu, Xin-Long; Zhang, Shi-Zhen; Sun, Wei; Li, Li-Jing; Wang, Han-Bing; Kong, Peng; Liu, Xiao-Qin; Liu, De-Min; Zhang, Xiao-Lan; Liu, Ya-Bin; Han, Mei

doi:10.1038/s41467-025-67707-3

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Published: 14 December 2025

Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer

Wen-Di Zhang^1,2,3^na1,
Dan-Dan Zhang^1,2,3^na1,
Xu Wang⁴^na1,
Chen-Yang Li⁵,
Fan-Qin Li^1,2,3,
Xin-Long Liu^1,2,3,
Shi-Zhen Zhang⁶,
Wei Sun⁶,
Li-Jing Li^1,2,3,
Han-Bing Wang⁷,
Peng Kong^1,2,3,
Xiao-Qin Liu^1,2,3,
De-Min Liu⁶,
Xiao-Lan Zhang⁵,
Ya-Bin Liu⁷ &
…
Mei Han ORCID: orcid.org/0000-0002-5338-4875^1,2,3

Nature Communications volume 17, Article number: 976 (2026) Cite this article

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Abstract

Colonic smooth muscle cell (CSMC) hypertrophy and hyperplasia have been described in both human ulcerative colitis (UC) and animal models. The deletion of smooth muscle (SM) 22α induces the phenotypic switching of SMCs. Here, we report that Sm22α-deficient mice develop spontaneous colitis, which is characterized by radical S-adenosyl-methionine domain-containing 2 (RSAD2)-driven mitochondrial dysfunction and inflammation in CSMCs and ferroptosis in the colonic mucosa. Mechanistically, RSAD2 mediates YTH m⁶A RNA-binding protein 1 (YTHDF1) methylation and activation, thereby increasing the mRNA N6-methyladenosine (m⁶A) modification and translation of dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation in CSMCs. Inflammatory CSMC-derived mitochondrial extracellular vesicles trigger intestinal epithelial ferroptosis by inducing ROS production. The ablation of RSAD2 in mice with SMC-specific Sm22α knockout alleviates colitis severity in this experimental model. Importantly, increases in both RSAD2 expression and the ferroptotic signature are observed in serum and/or colonic samples from UC patients. Overall, this study shows a mitochondrial mechanism underlying the ability of dysfunctional smooth muscle to drive colitis and highlights the potential of targeting the RSAD2-YTHDF1 axis as an innovative therapeutic strategy for colitis.

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Introduction

Inflammatory bowel disease (IBD) is a chronic, relapsing, and remitting inflammatory disorder of the gut that includes two main phenotypes, Crohn’s disease (CD) and ulcerative colitis (UC), and significantly disrupts bowel function and patient quality of life¹. Known disease risk alleles highlight key pathways in the pathogenesis of IBD, including innate and adaptive immunity, gut barrier function, and pathogen sensing and responses. Epithelial cells, goblet cells, stroma and immune cells in the intestinal mucosa interact with each other or with the intestinal microbiota and the immune system, sending and receiving signals². Furthermore, an inappropriate and continuing mucosal immune response to commensal microbes in a genetically susceptible host is also involved in intestinal inflammation in the gut and IBD². However, the underlying genes at risk loci have not been mapped to their cells or pathways of action.

The gastrointestinal tract comprises an epithelium surrounded by layers of smooth muscle cells (SMCs). Fully mature SMCs have a unique ability to reversibly alter their phenotype from a contractile state to a synthetic state to respond to environmental cues³. Smooth muscle (SM) 22α, a marker of mature SMCs, is involved in actin filament assembly and stress fiber formation⁴, which are required for maintaining the differentiated phenotype of SMCs^5,6. Our group and others have demonstrated that disruption of SM22α in vascular SMCs enhances the vascular inflammatory response by inducing the phenotypic switching of SMCs^7,8. SMC hypertrophy (enlarged muscle cells) and hyperplasia (increased numbers of muscle fibers) have been described in the inflamed intestine in both human IBD and animal disease models and are being increasingly recognized as the main contributors to increased wall thickness in the fibrotic process of IBD⁹. Furthermore, aberrant SMC phenotypes, as environmental variables, may contribute to dysregulation of the mucosal immune and commensal ecosystem in the gut, ultimately initiating colitis. However, the importance of the smooth muscle component in IBD has been less investigated. This prompted us to ask whether and how SM22α disruption promotes intestinal inflammatory damage.

Radical S-adenosyl-L-methionine domain-containing 2 (RSAD2) is an interferon-stimulated gene that is renowned for its antiviral properties. RSAD2 is involved in complex interplay with mitochondria, and binding of the iron–sulfur (Fe–S) cluster is necessary for the involvement of RSAD2 in mitochondrial metabolism. RSAD2 influences the proliferation and development of immune cells and inflammation via different signaling pathways¹⁰. However, the function of RSAD2 has been shown to vary across studies, and knowledge of this topic is lacking. Although a previous study demonstrated that increasing RSAD2 expression in neutrophils suppressed intestinal inflammation in a mouse model of DSS-induced colitis^11,12, very little is known about the crucial role of RSAD2 in the pro-inflammatory phenotypic switching of SMCs.

An increasing number of studies have underscored the new roles of YTH N6-methyladenosine (m⁶A) RNA-binding protein 1 (YTHDF1) in the gut¹³. Although YTHDF1 is largely dispensable for intestinal homeostasis under physiological conditions, YTHDF1 is required for intestinal epithelial regeneration and tumorigenesis because it maintains stem cell traits by amplifying Wnt/β-catenin signaling at the translational level¹⁴. Ythdf1 mRNA is upregulated in ulcerative colitis (UC) patients¹⁵ and downregulated in both LPS-induced macrophages and the colons of IBD model mice, whereas the upregulation of YTHDF1 alleviates the inflammatory response¹⁶, suggesting that YTHDF1-mediated RNA methylation events are involved in IBD pathogenesis¹⁷. In the current study, we demonstrate that SM22α deletion activates the RSAD2-YTHDF1 axis, leading to mitochondrial fragmentation and a proinflammatory phenotype in colonic SMCs (CSMCs). Mitochondrial extracellular vesicles (MitoEVs) from CSMCs induce intestinal epithelial cell ferroptosis and colitis development.

Results

SM22α deletion leads to spontaneous colitis

We first confirmed that the expression of SM22α was restricted to the muscular layer of the intestinal wall of wild-type (WT) mice (Supplementary Fig. 1a). Three-month-old Sm22α knockout (Sm22α^−/−) mice exhibited signs of colitis. Weight loss was evident in these mice (Fig. 1a), which passed paste-like stools with high water contents (Supplementary Fig. 1b–d). Gross and microscopic examinations of Sm22α^−/− mice revealed shorter colons (Fig. 1b) and increased histological activity index (HAI) scores, indicating architectural distortion and variation in the size and shape of crypts (Fig. 1c). Moreover, compared with their WT littermates, goblet cell loss, inflammatory cell infiltration and intestinal epithelial apoptosis, which are hallmarks of IBD¹⁸, were obvious in Sm22α^-/− mice (Fig. 1c–e). Disrupted epithelial hypoxia (Fig. 1f) and increased plasma levels of FITC-conjugated dextran (Fig. 1g) indicated an increase in intestinal barrier permeability in Sm22α^−/− mice, which was accompanied by impaired peristaltic contractions (Fig. 1h and Supplementary Fig. 1e, f). At the molecular level, the expression of the proinflammatory cytokines Tnf and Il-1β significantly increased in the colonic tissues of Sm22α-deficient mice, whereas the level of the anti-inflammatory cytokine Il-10 decreased (Fig. 1i), indicating an inflammatory phenotype. In addition, Sm22α^−/− mice exhibited decreased proliferation and differentiation of intestinal epithelial cells without evidence of epithelial metaplasia (Fig. 1f and Supplementary Fig. 1g).

**Fig. 1: SM22α deficiency induces spontaneous colitis.**

To determine the role of CSMCs in colitis development, we generated SMC-specific Sm22α knockout (smSm22α^−/−) mice by crossing mice withloxP sites flanking Sm22α (Sm22α ^fl/fl mice) with SMC-specific Cre-expressing transgenic mice (myh11Cre mice). Similar colitis-related changes, including weight loss (Fig. 1j), shorter colons (Fig. 1k), elevated HAI scores (Fig. 1l), and goblet cell loss (Fig. 1m), were observed in 3-month-old smSm22α^−/− mice. Additionally, intestinal permeability and peristaltic contraction were impaired (Fig. 1n, o) in these mice compared with Sm22α^fl/fl mice, which was accompanied by increased inflammation (Fig. 1p).

To further investigate whether SM22α deletion in CSMCs plays a unique role in DSS-induced inflammation in mice, we administered DSS to induce colitis. Compared with Sm22α^fl/fl control mice, smSm22α^−/− mice had significantly shorter survival times (Fig. 2a), lower body weights (Fig. 2b), higher disease activity index (DAI) scores (Fig. 2c), shorter colons (Fig. 2d, e) and more severe tissue damage (Fig. 2f, g) 7d after the administration of DSS. Consistently, higher intestinal permeability and more severe peristaltic dysfunction and inflammation were observed in DSS-treated smSm22α^−/− mice than in control mice (Fig. 2h–j). Additionally, we found that DSS impaired the contractility of CSMCs cultured in vitro (Supplementary Fig. 1h, i). Notably, the length of the small intestines shortened and the intestinal transit times of the marker that was orally administered, extended in Sm22α^−/− mice compared with WT mice at baseline (Supplementary Fig. 2a-c). The small intestinal wall, especially the ileum, was significantly damaged with higher histological score in Sm22α^−/− mice (Supplementary Fig. 2d), suggesting the small intestinal dysfunction. Nevertheless, no difference in the colitis phenotype between male and female Sm22α^−/− mice was observed (Supplementary Fig. 2e–j). The rescue of SM22α expression by the administration of an adeno-associated virus 2/9 (AAV) carrying SM22α markedly ameliorated colitis phenotype in Sm22α^−/− mice, with less pronounced colon shortening, tissue damage, peristaltic dysfunction and inflammation (Supplementary Fig. 3a–g). Collectively, these results suggest that the loss of SM22α in CSMCs plays a crucial role in colitis development.

Fig. 2: smSm22α−/− mice are more susceptible to dextran sulfate sodium (DSS)-induced colitis. Sm22α fl/fl and smSm22α−/− mice were administered 2.5% DSS in drinking water for 7 days, followed by sample collection, n = 10 mice per group. — Fig. 2: smSm22α^−/− mice are more susceptible to dextran sulfate sodium (DSS)-induced colitis. *Sm22α* ^fl/fl and smSm22α^−/− mice were administered 2.5% DSS in drinking water for 7 days, followed by sample collection, n = 10 mice per group.

SM22α deficiency disrupts mitochondrial homeostasis in CSMCs

To elucidate the molecular mechanisms linking SM22α deficiency to colitis, we performed RNA-seq and microarray analyses on colonic smooth muscle tissues from WT and Sm22α^−/− mice. Transcriptomic analysis revealed 2,028 differentially expressed genes (DEGs), including 898 upregulated genes and 1,130 downregulated genes, in Sm22α^−/− tissues (Supplementary Fig. 4a), as validated by quantitative PCR (qPCR) (Supplementary Fig. 4b). Gene Ontology (GO) enrichment analysis of the differentially expressed genes revealed that the upregulated genes were significantly associated with immune activation, inflammatory responses, and osteogenic processes. In contrast, the downregulated genes were primarily enriched in negative regulation of ossification, glycolysis, and fatty acid transport, suggesting that the colonic SMCs in Sm22α^−/− mice undergo phenotypic switching characterized by proinflammatory and osteoblast-like transdifferentiation (Supplementary Fig. 4c). Notably, mitochondrial material transport, amino acid metabolism, oxidative phosphorylation, and ATP production regulation were significantly suppressed in the CSMCs of Sm22α^−/− mice (Fig. 3a).

Fig. 3: Mitochondrial dysfunction occurs in Sm22α−/− colonic smooth muscle cells (CSMCs). — **Fig. 3: Mitochondrial dysfunction occurs in *Sm22α*^−/− colonic smooth muscle cells (CSMCs).**

Our previous study demonstrated that SM22α deficiency impaired mitochondrial structure and function in uterine SMCs, leading to contractility defects and dystocia¹⁹. Consistent with these findings, electron microscopy revealed mitochondrial shrinkage, severe cristae loss, and membrane rupture in primary Sm22α^−/− CSMCs (Fig. 3b). We next evaluated mitochondrial morphology and discovered that mitochondrial fragmentation was substantially increased in Sm22α^−/− cells. The mitochondrial number, mean length, and network branch counts were significantly lower in Sm22α^−/− CSMCs than in WT controls (Fig. 3c, d). Moreover, mitochondrial dysfunction was further corroborated by elevated mitochondrial reactive oxygen species (mtROS) levels (Fig. 3c) and reduced mitochondrial DNA (mtDNA) copy numbers and ATP production (Fig. 3e, f). Notably, among mitochondrial fusion and fission markers, only dynamin-related protein 1 (DRP1) was markedly upregulated in Sm22α^−/− CSMCs (Fig. 3g, h), which is consistent with a significant increase in mitochondrial fragmentation. Treatment with Mdivi-1, a selective DRP1 inhibitor, mitigated mitochondrial fragmentation in Sm22α^−/− CSMCs (Supplementary Fig. 5a, b), suggesting that DRP1 plays an essential role in mitochondrial dysfunction in Sm22α^−/− CSMCs.

Our previous research demonstrated that TNF induced the epigenetic silencing of SM22α transcription via H3K27 methylation in the SM22α promoter region in vascular SMCs⁸ and that the disruption of SM22α further aggravated vascular inflammation²⁰, prompting us to explore whether the synergistic interaction of TNF stimulation and SM22α ablation impaired mitochondrial function in Sm22α^−/− CSMCs. In line with our hypothesis, stimulation of CSMCs with TNF induced mitochondrial fragmentation and mtROS production (Supplementary Fig. 6a, b) and led to mitochondrial dysfunction (Supplementary Fig. 6c, d), which were similar to the phenotypic outcomes of Sm22α^−/− CSMCs. Furthermore, TNF treatment significantly increased DRP1 protein levels (Supplementary Fig. 6e, f). Notably, TNF treatment exacerbated mitochondrial fragmentation and dysfunction in Sm22α^−/− CSMCs (Supplementary Fig. 6g–l). Overall, these data indicate that inflammation signaling contributes to mitochondrial damage through the upregulation of DRP1 in CSMCs.

RSAD2 expression is upregulated during inflammation

To further elucidate the molecular mechanisms underlying mitochondrial dysfunction in Sm22α^−/− CSMCs, we compared our RNA-seq data with 1,218 mitochondria-related genes cataloged in the MitoCarta 3.0 database²¹. This analysis revealed 43 overlapping genes, 9 of which were upregulated and 34 of which were downregulated in Sm22α^−/− smooth muscle tissues (Fig. 4a, b). Notably, RSAD2, a free radical S-adenosylmethionine (SAM) enzyme associated with immune, inflammatory, and mitochondrial processes²², was significantly upregulated (Fig. 4b), suggesting its potential role in mediating mitochondrial dysfunction linked to SM22α deletion. Therefore, we examined the expression and distribution of RSAD2 in WT and Sm22α^−/− colonic tissues and found that the mRNA and protein levels of RSAD2 were markedly increased throughout the entire intestinal wall (Fig. 4c–g), especially in the smooth muscle layers of Sm22α^−/− mice (Fig. 4f). Parallel observations in human samples revealed thickening of the colonic muscularis propria and increased RSAD2 expression in both smooth muscle tissues and serum from patients with UC compared with controls without colitis (Supplementary Tables 1 and 2 and Supplementary Fig. 7a–d). Overall, these results suggest that RSAD2 plays a critical role in colitis pathogenesis.

Fig. 4: RSAD2 expression is upregulated in Sm22α−/− colonic smooth muscle cells (CSMCs). — **Fig. 4: RSAD2 expression is upregulated in *Sm22α*^−/− colonic smooth muscle cells (CSMCs).**

Next, we extended these findings by performing complementary experiments in CSMCs. Consistent with the in vivo observations, the expression of RSAD2 at both the mRNA and protein levels was significantly elevated (Fig. 4h–j), and RSAD2 colocalized mainly with mitochondria in the cytoplasm of Sm22α^−/− CSMCs (Fig. 4k, l). Sm22α knockdown by a specific siRNA increased the expression of the RSAD2 protein (Fig. 4m, n), whereas rescuing Sm22α expression in Sm22α^−/− CSMCs abolished the increase in RSAD2 protein expression (Fig. 4o, p). Consistently, TNF treatment significantly increased RSAD2 protein levels in CSMCs (Fig. 4q, r). Together, these results indicated that inflammatory signaling contributes to RSAD2 upregulation.

RSAD2 leads to mitochondrial fragmentation and dysfunction in CSMCs

RSAD2 is an endoplasmic reticulum (ER)-localized IFN-inducible iron sulfur (Fe–S) cluster-binding antiviral protein²². We next investigated whether RSAD2 is involved in mitochondrial dysfunction in CSMCs. Analysis of mitochondrial morphology revealed that mitochondrial fragmentation was induced by the overexpression of RSAD2 and that the majority of mitochondria were relatively short and punctate (Fig. 5a, b). Furthermore, mtROS levels markedly increased (Fig. 5a), which was accompanied by decreases in mtDNA copy numbers (Fig. 5c) and ATP production (Fig. 5d). Given the significant upregulation of DRP1 in Sm22α^−/− CSMCs or upon TNF treatment, we examined the effect of RSAD2 on DRP1 expression. RSAD2 overexpression markedly increased the protein level of DRP1 in CSMCs (Fig. 5e, f), indicating that RSAD2 strongly affects mitochondrial dysfunction.

**Fig. 5: RSAD2 promotes DRP1 expression and mitochondrial fragmentation.**

To further confirm the role of RSAD2 in mitochondrial dysfunction under inflammatory conditions, WT and Rsad2^−/− CSMCs were treated with TNF. We showed that RSAD2 deficiency mitigated TNF-induced mitochondrial dysfunction, which was characterized by reduced mitochondrial fragmentation (Fig. 5g, h) and decreased mtROS levels (Fig. 5g). Additionally, compared with those in WT cells, the mtDNA copy number and ATP production in Rsad2^−/− CSMCs increased upon TNF treatment (Fig. 5i, j). Consistent with these findings, DRP1 expression was significantly downregulated in Rsad2^−/− CSMCs compared with WT controls following TNF treatment (Fig. 5k, l), further indicating that RSAD2 plays a dominant role in DRP1-related mitochondrial dysfunction.

We next investigated whether RSAD2 deficiency plays a unique role in protecting mice against DSS-induced colitis. WT and Rsad2^−/− mice were given 2.5% DSS in their drinking water for 7 days. Compared with WT mice, Rsad2^−/− mice exhibited significantly less weight loss, lower DAIs, and less colon shortening (Supplementary Fig. 8a–d) after DSS administration, findings that were confirmed by histological analysis (Supplementary Fig. 8e–g). Furthermore, DSS-treated Rsad2^−/− mice survived longer (Supplementary Fig. 8h). Overall, our data support the conclusion that RSAD2 plays a crucial role in intestinal inflammation.

RSAD2 facilitates DRP1 translation through m⁶A deposition

To elucidate the molecular mechanism through which RSAD2 affects DRP1-related mitochondrial dysfunction, we examined the regulatory pathways involved in DRP1 protein expression, including mRNA transcription, stability, and translation, as well as ubiquitination and degradation, in CSMCs from WT and Rsad2^−/− mice. We showed that the mRNA level of Drp1 did not change upon TNF treatment (Fig. 6a), and its decay kinetics did not significantly differ between WT and Rsad2^−/− CSMCs upon actinomycin D treatment (Fig. 6b), suggesting that RSAD2 does not affect Drp1 mRNA transcription or stability. We then assessed the role of RSAD2 in DRP1 protein stability using ubiquitin immunoprecipitation assays. No significant differences in DRP1 ubiquitination or degradation were detected between WT and Rsad2^−/− CSMCs (Fig. 6c), effectively ruling out protein stability as a regulatory mechanism. Taken together, these results indicated that RSAD2 may increase DRP1 expression at the translational level.

Fig. 6: RSAD2 promotes Drp1 mRNA translation by facilitating the activity of the m6A reader YTHDF1. — **Fig. 6: RSAD2 promotes *Drp1* mRNA translation by facilitating the activity of the m⁶A reader YTHDF1.**

The N6-methyladenosine (m⁶A) RNA modification plays a crucial role in regulating RNA metabolism, including its transport, decay, and translation. Given that RNA methylation activity is associated with RSAD2²³, we hypothesized that RSAD2 influences Drp1 mRNA translation via m⁶A deposition. Under inflammatory conditions, global m⁶A levels were elevated in CSMCs, whereas RSAD2 deficiency significantly attenuated m⁶A deposition (Fig. 6d). Methylated RNA immunoprecipitation (MeRIP)–qPCR analysis revealed significant enrichment of m⁶A on Drp1 mRNA in CSMCs following TNF treatment (Fig. 6e). In contrast, Rsad2^−/− CSMCs exhibited markedly reduced m⁶A deposition under the same conditions, suggesting that RSAD2 modulates m⁶A deposition. However, unexpectedly, we did not detect an interaction between RSAD2 and Drp1 mRNA (Fig. 6f), which excluded the possibility that the enrichment of m⁶A on Drp1 mRNA was due to RSAD2-associated RNA methylation activity.

Next, we conducted RNA interference (RNAi) targeting key m⁶A regulatory proteins, including writers (METTL3 and METTL14), erasers (ALKBH5 and FTO), and readers (YTHDF1 and YTHDF3). Among these, knockdown of YTH domain family protein 1 (YTHDF1) significantly reduced m⁶A enrichment on the mRNA of Drp1 upon TNF treatment, as indicated by MeRIP–qPCR (Fig. 6g, h). m⁶A was recently shown to mediate the regulation of gene expression at the mRNA level through the direct recruitment of reader proteins to the transcript²⁴. We showed that the expression of the DRP1 protein decreased after YTHDF1 knockdown (Fig. 6i), suggesting that YTHDF1, as a reader, may increase the translation of Drp1 mRNA by recognizing and binding to its m⁶A modifications.

To verify our speculation, we investigated the binding of YTHDF1 to Drp1 mRNA via RNA immunoprecipitation (RIP) assays with an anti-YTHDF1 antibody. TNF treatment increased the interaction of YTHDF1 with Drp1 mRNA. Drp1 mRNA was strongly enriched in the immunoprecipitated complexes (Fig. 6j). RNA pull-down assays confirmed that YTHDF1 binds to Drp1 mRNA (Fig. 6k), which is consistent with the increase in DRP1 protein expression. Importantly, the binding of YTHDF1 to Drp1 mRNA was markedly suppressed in Rsad2^−/− CSMCs (Fig. 6j, k), indicating that YTHDF1 reader activity depends on RSAD2. Together, these data suggest that RSAD2 increases the translation of Drp1 mRNA through YTHDF1-mediated recognition of m⁶A modification.

RSAD2 mediates YTHDF1 methylation and activation

To determine how RSAD2 modulates YTHDF1 activity, we conducted computational docking analyses using the HDOCK server (Supplementary Fig. 9a). The top-scoring model predicted an interaction between RSAD2 and YTHDF1 (Supplementary Data 1). This interaction was validated by coimmunoprecipitation (CoIP) assays; furthermore, the binding interaction was enhanced following TNF treatment (Fig. 7a–c). Notably, the protein level of YTHDF1 did not change upon TNF treatment or RSAD2 deletion (Fig. 7d).

**Fig. 7: RSAD2 induces DRP1 protein expression through methylating YTHDF1.**

Posttranslational modifications of readers may also alter their m⁶A binding affinity, cellular localization and interactions with protein partners²⁵. Although RSAD2 is a radical SAM enzyme, neither the reaction it catalyzes nor its substrate has been identified²³. We hypothesized that RSAD2 may mediate YTHDF1 methylation. To validate the physical and functional interaction of RSAD2 with YTHDF1, we performed immunoprecipitation (IP) assays with anti-α-mono- and anti-dimethyl lysine antibodies. Western blot analysis revealed that YTHDF1 was indeed methylated in CSMCs (Fig. 7e). Furthermore, TNF treatment increased YTHDF1 methylation, which was abolished by RSAD2 deletion (Fig. 7f). These results have been also validated in vivo. In DSS-induced colitis models, YTHDF1 methylation was increased in smSm22α^−/− mice but reduced in Rsad2^−/− mice (Fig. 7g, h). Overall, these data indicated that RSAD2 plays a dominant role in YTHDF1 methylation.

To further define the role of RSAD2 in YTHDF1 methylation, we generated a series of RSAD2 truncation mutants, including the SAM domain (GFP-SAM) and a truncated SAM domain (GFP-ΔSAM) (Fig. 7i). Proximity ligation assays (PLAs) in HEK293A cells co-transfected with the HA-YTHDF1 and RSAD2 constructs revealed that both full-length (FL) RSAD2 and its SAM domain interacted with YTHDF1, whereas the SAM domain deletion construct (GFP-ΔSAM) failed to bind (Fig. 7j). CoIP assays further confirmed that the SAM domain mediated the binding of RSAD2 to YTHDF1 (Fig. 7k). Consistently, both full-length RSAD2 and the SAM domain increased YTHDF1 methylation (Fig. 7l). We validated these results in HEK293A cells co-transfected with the HA-YTHDF1 and GFP-SAM constructs, which exhibited strong interactions between the SAM domain and YTHDF1 (Fig. 7m), accompanied by increased YTHDF1 methylation (Fig. 7n). Moreover, overexpression of GFP-SAM markedly increased the effect of YTHDF1 on Drp1 mRNA expression (Fig. 7o), which was accompanied by an increase in DRP1 protein expression in CSMCs (Fig. 7p), indicating that the RSAD2-YTHDF1 axis positively affects DRP1 expression. Together, these results suggest that YTHDF1 is an endogenous substrate of RSAD2.

In addition to recognizing m⁶A modifications, reader proteins may indirectly regulate m⁶A deposition. For example, m⁶A readers can compete with m⁶A demethylases for binding to m⁶A sites, resulting in varying degrees of m⁶A deposition²⁶. As mentioned above, upon TNF treatment, Drp1 mRNA m⁶A deposition increased in CSMCs but decreased in Rsad2^−/− cells (Fig. 6e). The results of the RNA pull-down experiments confirmed that the binding of ALKBH5, an m⁶A demethylase, to Drp1 mRNA markedly decreased with increased YTHDF1 expression in TNF-treated cells compared with control cells (Supplementary Fig. 10a, b). This decrease was reversed in Rsad2^−/− CSMCs, which exhibited increased enrichment of ALKBH5 on Drp1 mRNA, coincident with the reduced m⁶A levels of Drp1 mRNA, suggesting that YTHDF1 occupancy at the m⁶A site of Drp1 mRNA suppresses ALKBH5 demethylase activity. Taken together, these findings suggest that RSAD2 promotes the YTHDF1-driven translational upregulation of Drp1 mRNA by mediating YTHDF1 methylation.

Inflammatory CSMC-derived mitochondrial extracellular vesicles induce ferroptosis in intestinal epithelial cells, contributing to colitis pathology

Recent advances have illustrated the functional importance of mitochondrial extracellular vesicle (MitoEV) release in an increasing number of tissue contexts²⁷. Specific mitochondrial stresses or inflammatory signals trigger MitoEV release²⁸. To verify the causal relationship between CSMC dysfunction and colitis, we first identified CD63-marked MitoEVs using immunofluorescence analysis in CSMCs (Supplementary Fig. 11a). We then isolated the MitoEVs from the conditioned medium of the CSMCs using differential centrifugation (Fig. 8a). Western blot analysis confirmed the presence of the mitochondrial outer membrane proteins TOM20 and TOM40 as well as the EV marker CD63 in CSMCs and in their released MitoEVs (Fig. 8b). Using FACS, we revealed that TNF treatment increased MitoEV release from CSMCs, which was significantly reduced in Rsad2^−/− cells (Supplementary Fig. 11b). To trace MitoEV-mediated mitochondrial transfer, we first labeled the mitochondria in CSMCs with MitoTracker Deep Red and the mitochondria in intestinal epithelial cells (IECs) with MitoTracker Green. MitoEVs isolated from the conditioned medium of TNF-treated CSMCs were then used to treat IECs (HCT116 cells). Immunofluorescence analysis confirmed that the CSMC-derived MitoEVs were taken up by IECs, a process inhibited by dynasore, a clathrin-mediated endocytosis inhibitor (Fig. 8c), suggesting that intercellular mitochondrial transfer occurred between CSMCs and IECs.

**Fig. 8: Mitochondrial extracellular vesicles (MitoEVs) secreted from CSMCs induce intestinal epithelial cells (IEC) ferroptosis.**

Intercellular mitochondrial transfer not only rescues tissue damage but also contributes to multifunctional cellular activity and the regulation of inflammation²⁹. An elegant study demonstrated that MitoEV-mediated mitochondrial transfer between cells in vivo resulted in increased mitochondrial ROS production in recipient cells³⁰. To examine this possibility, we treated IECs with MitoEVs derived from different cellular contexts, including WT and Rsad2^−/− CSMCs with or without TNF treatment. We observed increased lipid peroxidation (Fig. 8d, e) and elevated MDA levels (Fig. 8f) in IECs treated with MitoEVs of TNF-induced CSMCs, accompanied by a decreased GSH/GSSG ratio (Fig. 8g), suggesting increased oxidative stress. Similar results were observed in other colonic epithelial cell lines, including FHC and Caco-2 (Supplementary Fig. 11c, d). In contrast, Rsad2^−/− MitoEVs seemed to have a smaller effect on lipid ROS levels (Fig. 8d–g). Together, these results suggested that MitoEVs from inflammatory CSMCs induced lipid peroxidation in the intestinal epithelium.

Lipid peroxidation products are the main drivers of ferroptosis, an iron-dependent form of necrotic cell death³¹. We showed that TNF-induced MitoEVs increased the levels of total ferrous iron and free Fe²⁺ in IECs (Fig. 8h–j). Moreover, expression of the ferroptosis marker COX2 significantly increased while GPX4 expression was decreased (Fig. 8k). In contrast, inflammatory Rsad2^−/− MitoEVs had a mild effect on ferroptosis in IECs. In vivo, DSS-treated Rsad2^−/− mice displayed less epithelial cell ferroptosis than the WT controls did (Fig. 8l, m and Supplementary Fig. 8i–k).

Similar to the findings in IECs, mouse intestinal organoids were shown to internalize CSMC-derived MitoEVs (Supplementary Fig. 12a, b). Exposure to inflammatory MitoEVs resulted in reduced expression of the barrier-associated protein Claudin-1 in colonic organoids, which was accompanied by elevated oxidative stress and the pronounced downregulation of the ferroptosis marker protein GPX4 and the upregulation of COX2 (Supplementary Fig. 12c–e). In contrast, inflammatory Rsad2^−/− MitoEVs failed to induce substantial changes in ROS levels or ferroptosis markers in intestinal organoids (Supplementary Fig. 12c–e).

Given that RSAD2 expression is increased in Sm22α^−/− mice with colitis, we investigated whether ferroptosis occurs in the intestinal mucosa in vivo. Western blot analysis revealed significant upregulation of COX2, which was accompanied by the downregulation of GPX4 (Supplementary Fig. 13a, b). Immunofluorescence analysis corroborated the increase in COX2 expression and the reduction in GPX4 expression (Supplementary Fig. 13c). Moreover, ferrous ion and MDA assays confirmed the occurrence of ferroptosis in the intestinal mucosa of Sm22α^−/− mice (Supplementary Fig. 13d, e). Intriguingly, genetic deletion of RSAD2 in SMC-specific Sm22α knockout mice alleviated colitis severity (Supplementary Fig. 13f–h) and reduced epithelial ferroptosis (Supplementary Fig. 13i–m). Importantly, compared with those from healthy controls, the colonic mucosa from patients with UC exhibited a similar ferroptotic signature, with COX2 upregulation and GPX4 downregulation (Supplementary Fig. 7e). Together, these results further demonstrated that CSMC dysfunction caused intestinal epithelial damage.

Discussion

This study reveals the pivotal role of CSMC dysfunction in colonic inflammation through intercellular mitochondrial transfer and intestinal epithelial cell ferroptosis. We showed that SM22α deficiency-induced inflammation markedly increased the expression of RSAD2 in CSMCs, which increased the m⁶A modification of Drp1 mRNA via YTHDF1 methylation. This modification facilitates the translation of Drp1 mRNA, resulting in excessive mitochondrial fragmentation and CSMC functional impairment. Dysfunctional mitochondria are subsequently released from CSMCs via MitoEV and engulfed by IECs, triggering oxidative stress, ferroptosis, and epithelial injury and culminating in colitis.

In addition to being involved in motor function, SMCs that undergo phenotypic switching can synthesize and secrete proinflammatory cytokines, chemotaxis-associated molecules, and growth factors, which have been studied in detail in vascular muscle³². SMC plasticity likely also occurs in the gut³³ and could have important implications for the pathogenesis of different gut motor disorders. The smooth muscle hypertrophy and hyperplasia observed in both human IBD and animal disease models are generally considered to result from intestinal inflammation, but little is known about the ability of SMCs to modulate intestinal epithelial function. In the current study, we showed that the colonic SMCs of smSm22α^−/− mice underwent phenotypic switching are proinflammatory and exhibit osteoblast-like transdifferentiation. DSS-treated smSm22α^−/− mice displayed more tissue damage and a higher pathological score, further suggesting the involvement of SMC phenotypic switching in colitis pathogenesis.

RSAD2 (also known as Viperin, cig5, or vig1) is localized to the ER and is an interferon (IFN)-inducible multifunctional protein in various cell types³⁴. RSAD2 is constitutively expressed at high levels in the liver, heart, adipose tissue, and some immune cells. Research has shown that RSAD2 expression in the immune system can be directly or indirectly induced by various stimuli, including viral infection, interferons, synthetic analogs of double-stranded RNA or B-form DNA, and LPS, via IFN-dependent and IFN-independent pathways³⁵. The basal and IFN-induced expression of RSAD2 is negatively regulated by a multiexonic nucleus-localized long noncoding RNA known as lncRNA-CMPK2 (cytidylate monophosphate kinase 2)³⁶. IFN-independent induction of RSAD2 expression can occur through interferon regulatory factor 3 (IRF3), activator protein 1 (AP-1), and IRF1³⁵. In addition to its broad-spectrum antiviral activity³⁷, RSAD2 also regulates cellular lipid metabolism³⁸, mediates Toll-like receptor signaling pathways³⁹, and plays broader roles in cellular function and proliferation⁴⁰. Additionally, RSAD2 is localized to mitochondria in brown adipose tissue cells⁴¹ and transiently in human foreskin fibroblasts, where it regulates the β-oxidation of fatty acids and energy production³⁸. A recent study demonstrated that IFNλ can modulate the function of neutrophils by upregulating RSAD2, leading to the suppression of intestinal inflammation in a mouse model of DSS-induced colitis, which was recapitulated in human neutrophils^11,12. Here, we provide contradictory evidence concerning the role of RSAD2 in DSS-induced colitis. Increased expression of RSAD2 was observed in the colonic muscle layer of mice with colitis, colonic samples from UC patients, and CSMCs that had undergone proinflammatory phenotypic switching. RSAD2 contributed to mitochondrial fragmentation and dysfunction in CSMCs by increasing DRP1 expression and activity in a YTHDF1-dependent manner. Furthermore, after treatment with DSS, the colon shortening, tissue damage, weight loss, and disease activity index scores of Rsad2^−/− mice significantly decreased. We speculated that the positive or negative effects of RSAD2 on colitis may be associated with cell type. Together, our findings provide evidence for the cell type-specific function of RSAD2 and show that increased expression of RSAD2 in CSMCs plays a dominant role in the development of colitis.

YTHDF1, a critical m⁶A reader protein, plays a key role in the increased translation of m⁶A-modified mRNAs. YTHDF1 promotes the translation of target transcripts, and this activity is regulated by its glycan modification and modification of its partner RBP Fragile X mental retardation protein (FMRP)⁴². YTHDF1 activity is also regulated by other posttranslational modifications, such as O-GlcNAc glycosylation⁴³, ubiquitination⁴⁴, and lactylation⁴⁵. Furthermore, increased YTHDF1 expression has been demonstrated to be associated with disruption of intestinal barrier integrity in CD patients and colitis mice⁴⁶. Deletion of YTHDF1 induces CD8⁺ T cells to stimulate the production of high levels of programmed cell death 1 ligand 1, ultimately exacerbating IBD⁴⁷. YTHDF1 overexpression increases the mRNA expression of immunosuppressive molecules in an m⁶A-dependent manner and restructures the environment of inflammation in colitis⁴⁸. Additionally, the increase in DNA copy number and the overexpression of YTHDF1 are considered frequent events in UC and inflammatory colorectal cancer^49,50. Our study demonstrated a methylation-dependent mechanism of YTHDF1 activity in IBD pathogenesis. We revealed that RSAD2 directly interacted with YTHDF1 via its SAM domain, promoting the lysine methylation of YTHDF1, which was accompanied by increased YTHDF1-driven translation efficiency of m⁶A-modified Drp1 mRNA, contributing to SMC-driven intestinal epithelial inflammation. Further investigations are needed to determine whether targeting YTHDF1 may increase the efficacy of current IBD therapeutic regimens.

Given that the substrate of RSAD2 is not a protein but a common metabolite²³, we speculated that YTHDF1 methylation may not be directly catalyzed by the radical SAM activity of RSAD2 but may indirectly result from SAM accumulation. The binding of YTHDF1 to RSAD2 might lead to the inability of RSAD2 to catalyze the reductive cleavage of SAM⁵¹, resulting in SAM accumulation. In fact, both the accumulation of SAM and the exhaustion of the cellular pool of this metabolite affect the SAM-dependent methyl transferases of DNA, protein, and phospholipids, which are involved in different cellular processes⁵². This discovery provides insights into the regulatory landscape of m⁶A readers and their posttranslational regulation under inflammatory conditions, offering promising avenues for therapeutic intervention.

The functions of mitochondria are not only associated with their metabolic roles in producing energy and generating essential metabolites but also linked to diverse processes, including cell signaling, the initiation of apoptosis, immune responses, and the regulation of cell proliferation and development⁵³. Emerging studies have revealed that cells can also export some of their mitochondria for delivery to recipient cells, contributing to both normal physiological processes and disease pathogenesis. The release and capture of extracellular MitoEVs as cargo constitute common mechanisms of intercellular mitochondrial transfer. These MitoEVs are marked with the tetraspanins CD63, CD9, and CD81, which are used to eliminate damaged mitochondrial components. MitoEVs enable intercellular communication with either beneficial or harmful effects on recipient cells, depending on the cellular source as well as the nature of the cargo and the originating stimulus⁵⁴. Brown adipocytes undergoing thermogenic stress eliminate damaged mitochondrial components via the formation of MitoEVs²⁷. Osteoblasts release MitoEVs containing fragmented mitochondria to promote osteoprogenitor cell differentiation into osteoblasts⁵⁵. Our current study revealed that TNF treatment increased MitoEV release from CSMCs, which was significantly reduced in Rsad2^−/− cells. Furthermore, CSMC-derived MitoEVs were taken up by IECs. The TNF-induced increase in the number of MitoEVs resulted in an increase in the total ferrous iron content; moreover, the expression of the ferroptosis marker COX2 increased in IECs while the expression of GPX4 decreased, and these effects were reduced in Rsad2^−/− MitoEV-treated IECs. Moreover, the induction of ferroptosis by inflammatory CSMC-derived MitoEVs was also observed in intestinal crypt organoids. In line with this finding, ferroptosis increased in cells in the intestinal mucosa of Sm22α^−/− mice but decreased in Rsad2^−/− mice. Overall, our data suggest that CSMCs with an inflammatory phenotype exacerbate intestinal inflammation via the induction of ferroptosis by MitoEVs.

In the present study, we cannot exclude the potential effects of vascular smooth muscle on the intestinal mucosa and disease phenotype via changes in gut vascular barrier function. Given the importance of the muscular component in IBD, a mouse model of IBD induced by colonic smooth muscle-specific inactivation of the Sm22α gene is necessary in future studies. How RSAD2 expression is induced in the CSMCs of the inflamed intestine in vivo or in vitro, and what intermediate molecule mediates RSAD2-driven YTHDF1 methylation, are unclear. Additionally, a more in-depth characterization of inflammatory CSMC-derived MitoEV biology is needed to enable the use of these cells as biomarkers in clinical studies.

In conclusion, this study reveals a pathophysiological mechanism linking smooth muscle dysfunction and intestinal epithelial damage, emphasizing the importance of mitochondrial dynamics-related CSMC dysfunction in the development of colitis. RSAD2 and its downstream effectors, including YTHDF1 and DRP1, may serve as biomarkers for colitis activity and severity, enabling more precise disease monitoring and facilitating personalized treatment.

Methods

Study approval

Approval for all the experimental procedures was granted by the Institutional Laboratory Animal Care and Use Committee of Hebei Medical University, Permit No. IACUC-Hebmu-P2023006.

Human samples

The study involving humans received approval from the Medical Ethics Committee of Hebei Medical University, and informed consent was obtained from all participants (2023-001). Between May 2023 and May 2024, colonic tissue and blood samples were collected via endoscopic biopsies at the Second Hospital of Hebei Medical University. UC was diagnosed by colonoscopy and pathological examination of colon tissue samples. This research was conducted with the voluntary participation of individuals who were not offered any compensation. Sex or gender information was not collected, and no sex or gender analysis was carried out.

Animals

All animal studies were carried out in accordance with the guidelines sanctioned by the Ethics Committee for Animal Experiments and the Institutional Animal Care and Use Committee at Hebei Medical University. The experimental procedures followed the guidelines set by Directive 2010/63/EU of the European Parliament regarding the protection of animals used in scientific research. Cyagen Biosciences (S-KO-11081) provided C57BL/6 J wild-type (WT) mice and Rsad2^−/− mice. Sm22α^−/− mice were acquired from the Jackson Laboratory (006878), and Myh11-cre/ER^T2 mice were obtained from Youdu Bio (YDS0815). All the mice were kept in a pathogen-free facility under controlled conditions, including a temperature ranging from 21 to 23 °C, a humidity ranging from 50 to 60%, and a 12-h light/dark cycle. Mice aged 12–16 weeks were used for the experiments. Tamoxifen was administered intraperitoneally (i.p.) at 100 mg kg⁻¹ in corn oil, either as a single dose or every other day for 14 days, starting at 5–8 weeks of age.

Culture and treatment of CSMCs

After being isolated from the colonic muscular layer of young mice, the CSMCs were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) supplemented with 20% fetal bovine serum (FBS; Gibco) and 1% penicillin‒streptomycin (PS; Biological Industries). The cells were incubated in a humidified environment at 37 °C with 5% CO₂. Only cells from passages 4 through 10 were used in the experiments. The cells were serum-starved by incubation in 0.5% FBS for 24 h before being stimulated with TNF (10 ng mL⁻¹) and transfected with siRNA or plasmids.

Culture and treatment of intestinal organoids

Organoid cultures and crypt cells were isolated using methods detailed in earlier studies⁵⁶. The procedures involved dissecting the colon lengthwise, washing it with cold DPBS, and incubating the crypts in Gentle Cell Dissociation Reagent (STEMCELL Technologies) at room temperature on a rocking platform at 20 rpm for 20 min. A 70-μm cell strainer was used to filter the crypt suspension, and the crypt fraction was collected by centrifugation at 290 × g for 5 minutes at 4 °C. The crypt pellets were then embedded in Matrigel (BD Biosciences) on ice and subsequently plated in 24-well plates. Once Matrigel polymerization was complete, 750 μL of complete IntestiCult Organoid Growth Medium (STEMCELL Technologies) was added to each well at room temperature. The culture medium was changed every 2 days. The mitoEVs from the CSMCs were used to treat the organoids for 48 h prior to additional experiments.

Methylated RNA immunoprecipitation (MeRIP) assay

In accordance with the manufacturer’s protocol, the m⁶A modification level of the Drp1 mRNA was determined using an EpiQuik CUT & RUN m⁶A RNA Enrichment Kit (P-9018; Epigentek). Briefly, m⁶A-containing RNA fragments were selectively isolated using beads conjugated with an anti-m⁶A capture antibody. The alterations in m6A methylation of Drp1 mRNA were quantified by qPCR.

Plasmid or siRNA transfection

The cultured CSMCs were grown to 50–60% cofluence and then transfected with overexpression plasmid (General Biosystems, Anhui, China) or siRNA (Gene Pharma, Suzhou, China) using HighGene transfection reagent (ABclonal, Wuhan, China) according to the manufacturer’s instructions. All plasmids were constructed using standard methods and confirmed by DNA sequencing. The siRNA sequences used for siRNA transfection are listed in Supplementary Table.

Statistical analysis

Unless specified in the figure legends, the data are displayed as the means ± SEMs. Statistical analyses were performed using GraphPad Prism (version 10.1.2; GraphPad Software, La Jolla, CA, USA). Comparisons between two groups were evaluated using two-tailed Student’s t tests. For analyses involving more than two groups, one-way ANOVA followed by Tukey’s post hoc multiple comparisons test was applied. The data and images represent at least three independent experiments. Statistical significance was defined as P < 0.05.

The Online Data Supplement contains an expanded methods section.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under accession code PRJNA1312247(ID 1312247 - BioProject - NCBI). All other data are available in the article and its Supplementary files. Source data are provided with this paper.

References

Ramos, G. P. & Papadakis, K. A. Mechanisms of disease: inflammatory bowel diseases. Mayo Clin. Proc. 94, 155–165 (2019).
Article CAS PubMed Google Scholar
Khor, B., Gardet, A. & Xavier, R. J. Genetics and pathogenesis of inflammatory bowel disease. Nature 474, 307–317 (2011).
Article ADS CAS PubMed PubMed Central Google Scholar
Scirocco, A. et al. Cellular and molecular mechanisms of phenotypic switch in gastrointestinal smooth muscle. J. Cell Physiol. 231, 295–302 (2016).
Article CAS PubMed Google Scholar
Zhang, J. C. et al. Analysis of SM22alpha-deficient mice reveals unanticipated insights into smooth muscle cell differentiation and function. Mol. Cell. Biol. 21, 1336–1344 (2001).
Article CAS PubMed PubMed Central Google Scholar
Fu, Y. et al. Mutagenesis analysis of human SM22: characterization of actin binding. J. Appl. Physiol. 89, 1985–1990 (1985).
Han, M. et al. Smooth muscle 22 alpha maintains the differentiated phenotype of vascular smooth muscle cells by inducing filamentous actin bundling. Life Sci. 84, 394–401 (2009).
Article CAS PubMed Google Scholar
Shen, J. et al. Disruption of SM22 promotes inflammation after artery injury via nuclear factor kappaB activation. Circ. Res. 106, 1351–1362 (2010).
Article ADS CAS PubMed PubMed Central Google Scholar
Shu, Y. N. et al. CKII-SIRT1-SM22α loop evokes a self-limited inflammatory response in vascular smooth muscle cells. Cardiovasc. Res. 113, 1198–1207 (2017).
Article CAS PubMed Google Scholar
Speca, S., Giusti, I., Rieder, F. & Latella, G. Cellular and molecular mechanisms of intestinal fibrosis. World J. Gastroenterol. 18, 3635–3661 (2012).
Article CAS PubMed PubMed Central Google Scholar
Chen, S. et al. Crucial roles of RSAD2/viperin in immunomodulation, mitochondrial metabolism and autoimmune diseases. Inflammation 48, 520–540 (2025).
Article CAS PubMed Google Scholar
Broggi, A., Tan, Y., Granucci, F. & Zanoni, I. IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function. Nat. Immunol. 18, 1084–1093 (2017).
Article CAS PubMed PubMed Central Google Scholar
Starling, S. Neutrophils: interfering with intestinal inflammation. Nat. Rev. Immunol. 17, 594 (2017).
Article CAS PubMed Google Scholar
Wang, H., Han, J. & Zhang, X. A. Interplay of m6A RNA methylation and gut microbiota in modulating gut injury. Gut Microbes 17, 2467213 (2025).
Article PubMed PubMed Central Google Scholar
Han, B. et al. YTHDF1-mediated translation amplifies Wnt-driven intestinal stemness. EMBO Rep. 21, e49229 (2020).
Article CAS PubMed PubMed Central Google Scholar
Motawi, T. K., Shaker, O. G., Amr, G. & Senousy, M. A. RNA methylation machinery and m(6)A target genes as circulating biomarkers of ulcerative colitis and Crohn’s disease: correlation with disease activity, location, and inflammatory cytokines. Clin. Chim. Acta 561, 119831 (2024).
Article CAS PubMed Google Scholar
Xu, X. et al. hucMSC-Ex alleviates inflammatory bowel disease in mice by enhancing M2-type macrophage polarization via the METTL3-Slc37a2-YTHDF1 axis. Cell Biol. Toxicol. 40, 74 (2024).
Article PubMed PubMed Central Google Scholar
Sebastian-delaCruz, M. et al. Implication of m6A mRNA methylation in susceptibility to inflammatory bowel disease. Epigenomes 4, 16 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kurashima, Y. & Kiyono, H. Mucosal ecological network of epithelium and immune cells for gut homeostasis and tissue healing. Annu. Rev. Immunol. 35, 119–147 (2017).
Article CAS PubMed Google Scholar
Gao, Y. et al. Smooth muscle 22α deficiency impairs oxytocin-induced uterine contractility in mice at full-term pregnancy. Biochem. Biophys. Res. Commun. 529, 884–889 (2020).
Article ADS CAS PubMed Google Scholar
Shu, Y. N. et al. SM22α inhibits vascular inflammation via stabilization of IκBα in vascular smooth muscle cells. J. Mol. Cell Cardiol. 84, 191–199 (2015).
Article ADS CAS PubMed Google Scholar
Rath, S. et al. MitoCarta3.0: an updated mitochondrial proteome now with sub-organelle localization and pathway annotations. Nucleic Acids Res. 49, D1541–D1547 (2021).
Article CAS PubMed Google Scholar
Seo, J. Y., Yaneva, R., Hinson, E. R. & Cresswell, P. Human cytomegalovirus directly induces the antiviral protein viperin to enhance infectivity. Science 332, 1093–1097 (2011).
Article ADS CAS PubMed Google Scholar
Fenwick, M. K., Li, Y., Cresswell, P., Modis, Y. & Ealick, S. E. Structural studies of viperin, an antiviral radical SAM enzyme. Proc. Natl. Acad. Sci. USA 114, 6806–6811 (2017).
Article ADS CAS PubMed PubMed Central Google Scholar
Liu, Y. et al. N (6)-methyladenosine RNA modification-mediated cellular metabolism rewiring inhibits viral replication. Science 365, 1171–1176 (2019).
Article ADS CAS PubMed Google Scholar
He, P. C. & He, C. m(6) A RNA methylation: from mechanisms to therapeutic potential. EMBO J. 40, e105977 (2021).
Article CAS PubMed PubMed Central Google Scholar
Wang, J. et al. ALKBH5 attenuates mitochondrial fission and ameliorates liver fibrosis by reducing Drp1 methylation. Pharmacol. Res. 187, 106608 (2023).
Article ADS CAS PubMed Google Scholar
Rosina, M. et al. Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue. Cell Metab. 34, 533–548.e12 (2022).
Article CAS PubMed PubMed Central Google Scholar
König, T. & McBride, H. M. Mitochondrial-derived vesicles in metabolism, disease, and aging. Cell Metab. 36, 21–35 (2024).
Article PubMed Google Scholar
Liu, D. et al. Intercellular mitochondrial transfer as a means of tissue revitalization. Signal Transduct. Target Ther. 6, 65 (2021).
Article PubMed PubMed Central Google Scholar
Brestoff, J. R. et al. Intercellular mitochondria transfer to macrophages regulates white adipose tissue homeostasis and is impaired in obesity. Cell Metab. 33, 270–282.e8 (2021).
Article CAS PubMed Google Scholar
Dixon, S. J. et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell 149, 1060–1072 (2012).
Article CAS PubMed PubMed Central Google Scholar
Owens, G. K., Kumar, M. S. & Wamhoff, B. R. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol. Rev. 84, 767–801 (2004).
Article CAS PubMed Google Scholar
Nair, D. G., Han, T. Y., Lourenssen, S. & Blennerhassett, M. G. Proliferation modulates intestinal smooth muscle phenotype in vitro and in colitis in vivo. Am. J. Physiol. Gastrointest. Liver Physiol. 300, G903–G913 (2011).
Article CAS PubMed Google Scholar
Seo, J. Y., Yaneva, R. & Cresswell, P. Viperin: a multifunctional, interferon-inducible protein that regulates virus replication. Cell Host Microbe 10, 534–539 (2011).
Article CAS PubMed PubMed Central Google Scholar
Rivera-Serrano, E. E. et al. Viperin reveals its true function. Annu. Rev. Virol. 7, 421–446 (2020).
Article CAS PubMed PubMed Central Google Scholar
Kambara, H. et al. Negative regulation of the interferon response by an interferon-induced long non-coding RNA. Nucleic Acids Res. 42, 10668–10680 (2014).
Article CAS PubMed PubMed Central Google Scholar
Rivieccio, M. A. et al. TLR3 ligation activates an antiviral response in human fetal astrocytes: a role for viperin/cig5. J. Immunol. 177, 4735–4741 (2006).
Article CAS PubMed Google Scholar
Seo, J. Y. & Cresswell, P. Viperin regulates cellular lipid metabolism during human cytomegalovirus infection. PLoS Pathog. 9, e1003497 (2013).
Article CAS PubMed PubMed Central Google Scholar
Saitoh, T. et al. Antiviral protein Viperin promotes Toll-like receptor 7- and Toll-like receptor 9-mediated type I interferon production in plasmacytoid dendritic cells. Immunity 34, 352–363 (2011).
Article CAS PubMed Google Scholar
Aguilar, H. et al. Biological reprogramming in acquired resistance to endocrine therapy of breast cancer. Oncogene 29, 6071–6083 (2010).
Article CAS PubMed Google Scholar
Eom, J. et al. Intrinsic expression of viperin regulates thermogenesis in adipose tissues. Proc. Natl. Acad. Sci. USA 116, 17419–17428 (2019).
Article ADS CAS PubMed PubMed Central Google Scholar
Zou, Z. & He, C. The YTHDF proteins display distinct cellular functions on m(6)A-modified RNA. Trends Biochem. Sci. 49, 611–621 (2024).
Article CAS PubMed PubMed Central Google Scholar
Chen, Y. et al. O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins. Nat. Cell Biol. 25, 1676–1690 (2023).
Article CAS PubMed PubMed Central Google Scholar
Wang, A. et al. USP47 inhibits m6A-dependent c-Myc translation to maintain regulatory T cell metabolic and functional homeostasis. J. Clin. Investig. 133, e169365 (2023).
Article CAS PubMed PubMed Central Google Scholar
Liu, B. et al. Severe fever with thrombocytopenia syndrome virus induces lactylation of m6A reader protein YTHDF1 to facilitate viral replication. EMBO Rep. 25, 5599–5619 (2024).
Article PubMed PubMed Central Google Scholar
Wang, J. et al. Elevated SPARC disrupts the intestinal barrier integrity in Crohn’s disease by interacting with OTUD4 and activating the MYD88/NF-κB pathway. Adv. Sci. 12, e2409419 (2025).
Article Google Scholar
Du, Y. et al. Role of epigenetic modifications and aging in inflammatory bowel disease. Medcomm Future Med. 2, e63 (2023).
Chen, Q. et al. WTAP/YTHDF1-mediated m6A modification amplifies IFN-γ-induced immunosuppressive properties of human MSCs. J. Adv. Res. 71, 441–455 (2025).
Article CAS PubMed Google Scholar
Gu, C. et al. Immune infiltration of Ulcerative Colitis and detection of the m6A subtype. J. Immunol. Res. 2022, 7280977 (2022).
Article PubMed PubMed Central Google Scholar
Wang, S. et al. N6-methyladenosine reader YTHDF1 promotes ARHGEF2 translation and RhoA signaling in colorectal cancer. Gastroenterology 162, 1183–1196 (2022).
Article CAS PubMed Google Scholar
Nelp, M. T., Young, A. P., Stepanski, B. M. & Bandarian, V. Human viperin causes radical SAM-dependent elongation of escherichia coli, hinting at its physiological role. Biochemistry 56, 3874–3876 (2017).
Article CAS PubMed Google Scholar
Honarmand Ebrahimi, K. A unifying view of the broad-spectrum antiviral activity of RSAD2 (viperin) based on its radical-SAM chemistry. Metallomics 10, 539–552 (2018).
Article CAS PubMed Google Scholar
Borcherding, N. & Brestoff, J. R. The power and potential of mitochondria transfer. Nature 623, 283–291 (2023).
Article ADS CAS PubMed PubMed Central Google Scholar
Picca, A. et al. Circulating extracellular vesicles: friends and foes in neurodegeneration. Neural Regen. Res. 17, 534–542 (2022).
Article CAS PubMed Google Scholar
Suh, J. et al. Mitochondrial fragmentation and donut formation enhance mitochondrial secretion to promote osteogenesis. Cell Metab. 35, 345–360.e7 (2023).
Article CAS PubMed Google Scholar
Clevers, H. Modeling development and disease with organoids. Cell 165, 1586–1597 (2016).
Article ADS CAS PubMed Google Scholar

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (32371161 to M.H., 82370474 to P.K., and 82400508 to D.D.Z.), the S&T Program of Hebei (22377759D to Y.B.L.), and the Graduate Student Innovation Fund of Hebei Province (CXZZBS2023101 to W.D.Z).

Author information

These authors contributed equally: Wen-Di Zhang, Dan-Dan Zhang, Xu Wang.

Authors and Affiliations

Department of Biochemistry and Molecular Biology, College of Basic Medicine, Hebei Medical University, Shijiazhuang, China
Wen-Di Zhang, Dan-Dan Zhang, Fan-Qin Li, Xin-Long Liu, Li-Jing Li, Peng Kong, Xiao-Qin Liu & Mei Han
Key Laboratory of Neural and Vascular Biology of Ministry of Education, Hebei Medical University, Shijiazhuang, China
Wen-Di Zhang, Dan-Dan Zhang, Fan-Qin Li, Xin-Long Liu, Li-Jing Li, Peng Kong, Xiao-Qin Liu & Mei Han
Key Laboratory of Vascular Biology of Hebei Province, Hebei Medical University, Shijiazhuang, China
Wen-Di Zhang, Dan-Dan Zhang, Fan-Qin Li, Xin-Long Liu, Li-Jing Li, Peng Kong, Xiao-Qin Liu & Mei Han
Department of Pathology, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China
Xu Wang
Department of Gastroenterology, the Second Hospital of Hebei Medical University, Shijiazhuang, China
Chen-Yang Li & Xiao-Lan Zhang
Department of Cardiology, the Second Hospital of Hebei Medical University, Shijiazhuang, China
Shi-Zhen Zhang, Wei Sun & De-Min Liu
Department of Gastrointestinal Surgery, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China
Han-Bing Wang & Ya-Bin Liu

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Contributions

M.H. designed the study. W.D.Z., X.W., D.D.Z., X.L.L., S.Z.Z., W.S., X.Q.L., L.J.L., H.B.W., and D.M.L. performed the animal study and analyzed the data. W.D.Z. and P.K. performed the sequencing experiments, bioinformatics analyses, and statistical analyses. X.L.Z., Y.B.L., and C.Y.L. collected the human samples and provided material support. D.D.Z. and F.Q.L. were responsible for visualization. W.D.Z. assisted with the drafting of the manuscript. M.H. is the principal investigator who obtained funding and drafted the manuscript. All the authors reviewed, contributed to, and approved the final manuscript.

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Zhang, WD., Zhang, DD., Wang, X. et al. Activation of the RSAD2-YTHDF1 axis in smooth muscle causes inflammatory bowel disease via intercellular mitochondrial transfer. Nat Commun 17, 976 (2026). https://doi.org/10.1038/s41467-025-67707-3

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Received: 26 February 2025
Accepted: 05 December 2025
Published: 14 December 2025
Version of record: 27 January 2026
DOI: https://doi.org/10.1038/s41467-025-67707-3