Targeting NAT10 alleviates colonic senescence and elderly-onset colitis by disrupting N4-acetylation of DYRK1A

Domain: chemistry
Published: 2026-03-01
Editorial outcome: minor_revision

Abstract
Intestinal aging characterized by imbalance between cell senescence and mucosal self-renewal, increases susceptibility to the elderly-onset ulcerative colitis (UC), while the underlying mechanisms remain elusive. Here, we identify mRNA N4-acetylcytidine (ac4C) modification and its specific writer, N-acetyltransferase 10 (NAT10), as critical regulators of human colonic epithelial cell senescence. Knockdown of NAT10 significantly alleviates human colonic epithelial cell senescence in vitro and colonoid and intestinal aging in vivo in aged mice. Using ac4C-modified transcriptome sequencing, we reveal that NAT10 stabilizes DYRK1A mRNA through ac4C modification, thereby driving colon epithelial senescence. Moreover, NAT10 and DYRK1A are markedly upregulated in ulcerative colitis tissues from elderly patients and positively correlate with disease severity. Knockdown of NAT10, treatment with Nat10 or Dyrk1a inhibitor, alleviates colitis in aged mice. Collectively, these findings suggest that modulating NAT10-mediated RNA ac4C modification could rejuvenate intestinal aging and provide a novel therapeutic strategy for elderly-onset colitis.

Introduction
Aging is characterized by progressive decline in organ physiology and cellular function, ultimately resulting in pathological alterations1,2. Aging-related intestinal changes include a thinner mucus layer, increased intestinal permeability, and impaired intestinal stem cell function3-5. Previous studies have demonstrated that aging-related intestinal deterioration contributes to the pathogenesis of colitis in aged rodents6-10. Consistently, recent epidemiological surveys reveal that ulcerative colitis (UC) exhibits a second incidence peak, with approximately 10% to 20% of cases occurring in individuals over 60 years old11,12. Treatment options for elderly UC patients are often constrained by comorbidities13,14. Additionally, elderly-onset UC patients frequently exhibit suboptimal responses to biologic therapies and face an increased risk of adverse reactions15,16. Beyond immunomodulators and biologic therapies, targeting intestinal aging represents a promising approach to mitigate elderly-onset UC17.

Intestinal epithelial cells (IECs) form a critical barrier that protects the body from environmental toxins and pathogens18. Cell senescence, defined by the permanent halt in cell proliferation and defective apoptosis, is a cellular response to various sublethal endogenous and exogenous stresses19. Numerous factors have been implicated in driving cell senescence, including impaired DNA repair, mitochondrial dysfunction, disruption of protein homeostasis, and alterations in epigenetic regulation20. Epigenetic represents a reversible mechanism links genotype to phenotype, which plays an important role in modulating the aging process in response to environmental stimulation21. Among them, RNA modifications represent an intriguing epitranscriptomic mechanism that fine-tunes gene expression, enabling rapid cellular responses and offering an energy-efficient means through the regulation of mRNA stability and translation. To date, over 170 types of RNA modifications have been identified, about 10 of which have been validated in mRNA within the mammalian transcriptome22. The ac4C modification of mRNA has been shown to regulate RNA stability and translation efficiency23. Recent studies highlight the significance of mRNA ac4C modifications in various biological processes, such as growth, development, and tumorigenesis24-26. However, the roles of mRNA ac4C modification in regulating cell senescence and age-related diseases remain largely unexplored.

Here, we reported a significant upregulation of mRNA ac4C modification and its unique known writer N-acetyltransferase 10 (NAT10), and uncovered their roles on colon epithelial cell senescence. NAT10 stabilizes DYRK1A mRNA, thereby promoting colon epithelial cell senescence in an RNA ac4C-dependent manner. Notably, Nat10/Dyrk1a axis plays a critical role in colonic senescence, and provides therapeutic targets to mitigate colitis in aged mice. Clinically, NAT10 and DYRK1A are upregulated in ulcerative colitis tissues from elderly patients, and related with more severe disease status. Collectively, these findings suggest that targeting NAT10-mediated RNA ac4C modification may rejuvenate colonic cell senescence and alleviate the elderly-onset UC.

Results
mRNA ac4C modification and its writer NAT10 are upregulated in the senescent colon epithelial cells

We establish H2O2- (H-Sen) or Doxorubicin- (DP-Sen) induced cell senescence models in the immortalized colon epithelial cell line NCM460 and the diploid finite colon epithelial cell line CCD841. We then utilized LC-MS/MSE to detect the mRNA modification from both above senescent and proliferative cells (Figure. 1a). The senescent state of the cells was confirmed by SA-Gal staining (Figure. 1b), G1 cell-cycle arrest (Supplementary Figure. 1a,b) and the upregulation of classical pro-inflammatory senescence-associated secretory phenotype (SASP) genes (IL-1β and IL-6) (Supplementary Figure. 1c,d). The ac4C modification of mRNA was consistently upregulated in both H-Sen and D-Sen induced senescent NCM460 and CCD841 cell lines (Figure. 1c,d). N-acetyltransferase 10 (NAT10), the specific writer of mRNA ac4C modification, were upregulated in senescent colon epithelial cells, accompanied by increased expression level of aging-related markers (p53, p21, p16 and γ-H2AX) and reduced expression of the cell-cycle regulator Cyclin D1 (Figure. 1e). We next established a replicative senescence model when passage 20 in CCD841 cells, characterized by an enlarged and flattened morphology and increased proportion of SA-Gal positive cells (Figure. 1f and Supplementary Figure. 1e,f). Similarly, the RNA ac4C modification and NAT10 protein levels was significantly upregulated in replicative-senescent CCD841 cells (Figure. 1g and Supplementary Figure. 1g).

NAT10 regulates colon epithelial cell senescence through mRNA ac4C modification

We then explore whether NAT10 regulates the senescence of colon epithelial cells in an RNA ac4C modification-dependent manner. Results showed that overexpression of wild type NAT10 (NAT10-WT) increased mRNA ac4C modification which was impaired by NAT10 knockdown (Supplementary Figure. 2a,b), whereas overexpression of mutant plasmid (NAT10-MUT) failed (Figure. 2a). Overexpression of NAT10-WT, while not NAT10-MUT, could partially reverse the downregulation of aging-related markers (p53, p21, p16 and γ-H2AX) and the percentages of SA-Gal and γ-H2AX positive cells, as well as upregulation of Cyclin D1 and the positive percentage of EdU under H2O2 or DORX treatment condition (Figure. 2b,c and Supplementary Figure. 2c-e).

Resistance to apoptosis is a hallmark feature of cellular senescence27. We next investigated the role of NAT10 in the senescent cell viability (Figure. 2d). A significant decrease number of colonies formation was observed in senescent colonic epithelial cells following 48 hours of NAT10 siRNA transfection. Overexpression of NAT10-WT, while not NAT10-MUT, increased the number of colonies in both H-Sen and D-Sen cells(Figure. 2e and Supplementary Figure. 2f). In addition, NAT10 knockdown reduced cell viability, but increased caspase 3/7 apoptotic activity in senescent cells. Reintroduction of NAT10-WT could rescue this phenotype (Figure. 2f,g and Supplementary Figure. 2g,h). Apoptosis assays further confirmed that NAT10 maintained the resistance of senescent cells to apoptosis in an RNA ac4C-dependent manner. (Figure. 2h and Supplementary Figure. 2i).

Knockdown Nat10 alleviates intestinal aging in mice

Previous findings on senescent colonic cells prompted us to further explore the expression of Nat10 in the colon of mice at different ages. Nat10 is predominantly localized in the colonic epithelial crypts, and its protein expression levels progressively increased in the colonic tissues in mice from 2, 6, 12, to18 months old (Figure. 3a,b). Accordingly, RNA ac4C modifications and the expression of Il-1β, Il-6, and TNF-α were upregulated in the intestines of 18-mon mice compared to those of 2-mon mice (Supplementary Figure. 3a,b).

To investigate the role of Nat10 in colons, Littermate Nat10+/- and Nat10-WT mice were co-housed, and colonic cell senescence was assessed in aged mice (Figure. 3c). In colonic tissues from Aged-Nat10+/- mice, Nat10 protein expression levels were significantly lower compared to Aged-WT mice. As marker of cell senescence, the expressions of p16, p21 and p53 were decreased, while Lgr5 and Ocln were upregulated in Aged-Nat10+/- mice (Figure. 3d). Furthermore, the colonic mucosal height and the fluorescence intensity of Ocln as well as the number of Ki67-positive and goblet cells (Muc2+) were increased in Aged-Nat10+/- while the number of γ-H2AX+ diminished (Figure. 3e and Supplementary Figure. 3c). Additionally, qPCR analysis showed that the expression of Il-1β, Il-6, and TNF-α in colonic tissues were significantly downregulated in Aged-Nat10+/- mice (Supplementary Figure. 3d). Similarly, knockdown of Nat10 via AAV-shNat10 delivery system in aged mice also resulted in decreased expression of p16 and p21(Figure. 3f), accompanied by increased expression of Ocln and Ki67⁺ in epithelial cells (Figure. 3g and Supplementary Figure. 3e). Collectively, these findings indicate that lowering NAT10 protein levels can diminish senescent cell accumulation and rejuvenate colon stem cells, thus enhancing mucosal barrier regeneration.

To further substantiate the role of NAT10 in intestinal aging, we intraperitoneally injected with a NAT10 inhibitor Remodelin, to aged mice every three days for two months (Supplementary Figure. 4a). Biomarker of p53 and p21 were significantly downregulated in colon tissues when treated with Remodelin (Supplementary Figure. 4b). A significant increase in colon mucosal height, goblet cells and Ki67 positive cells in crypts, as well as the fluorescence intensity of the Ocln was observed (Supplementary Figure. 4c,d). In addition, Remodelin exhibited well tolerability in aged mice, without significant alterations in serum biochemical parameters or histopathological change in major organs (heart, liver, spleen, lung, and kidney) (Supplementary Figure. 4e,f).

DYRK1A as a target gene of NAT10-mediated RNA ac4C modification

To further explore candidate target genes with ac4C modification, we performed acRIP-seq on senescent colon epithelial cell knockdown of NAT10 or not, as well as wild type NCM460 cells (Figure. 4a). A summary of peak numbers and ac4C-enriched gene counts for each sample is presented in the Supplementary Table 1. We observed that ac4C modifications on mRNA were enriched in CDS region (Figure. 4b and Supplementary Figure. 5a). Motif analysis showed that ac4C modifications were highly enriched within a C-rich sequence, which is consistent with previously reported results28 (Supplementary Figure. 5b). 1,228 hyperacetylated peaks (log₂FC > 0) corresponding to 858 genes were identified in the H-Sen group compared with the control group. In contrast, 640 hypoacetylated peaks (log₂FC<0) corresponding to 529 genes were detected in the H-Sen NAT10-Si group. GO enrichment analysis of hyperacetylated genes in senescent NCM460 cells revealed that these transcripts were functionally enriched in several senescence-related biological processes, including autophagy, type I interferon signaling, and DNA damage signaling27,29,30. While hypoacetylated genes in Nsenescent NCM460 cells with NAT10 knockdown showed that these genes were primarily involved in the transmission and regulation of DNA damage response signaling pathways (Figure. 4c).

To identify the potential targets through which NAT10-mediated RNA ac4C modification, we first identified 32 protein-coding transcripts that exhibited increased ac4C abundance (|log₂FC|>1.5) in H-Sen. Upon NAT10 knockdown in H-Sen (H-Sen-Si vs. H-Sen), 11 protein-coding transcripts showed reduced ac4C abundance (|log₂FC|>1.5). We intersected the differentially acetylated transcripts and identified four candidate genes: THAP5, DYRK1A, LMLN, and MVB12A (Figure. 4d). acRIP-qPCR confirmed that ac4C enrichment on DYRK1A and LMLN were consistent with the sequencing data (Figure. 4e). In line with this, qPCR analyses indicated that only DYRK1A mRNA showed consistent changes (Figure. 4f). It has been reported that NAT10 enhances mRNA acetylation, which subsequently increases mRNA stability and translation23,31. To further validate this, we performed RNA sequencing followed by transcription inhibitor actinomycin D (Act-D) treatment, and found that only DYRK1A mRNA, among the candidates (THAP5, DYRK1A, LMLN, and MVB12A), was regulated by NAT10, with its expression decreasing over time (Figure. 4g and Supplementary Figure. 5c). Consistently, merged IGV visualization for three groups of our acRIP-seq data revealed multiple changes in ac4C modifications across the CDS region of DYRK1A mRNA (Figure. 4h and Supplementary Figure. 5d). Western blot analysis further confirmed that NAT10 regulates DYRK1A protein expression (Figure. 4i). Collectively, these findings suggest that DYRK1A is a critical downstream effector of ac4C-mediated cellular senescence in colonic epithelial cells.

NAT10 promotes colon epithelial cell senescence by regulating DYRK1A mRNA stabilization

We observed significant enrichment of ac4C modification on the CDS domain of DYRK1A transcripts in senescent NCM460 cells (Figure. 5a). Additionally, the RNA and protein expression levels of DYRK1A were increased in senescent colon epithelial cells (Figure. 5b). Further immunofluorescence analysis confirmed that DYRK1A expression was relatively elevated in senescent cells, accompanied with higher DNA damage marker γ-H2AX (Figure. 5c). We hypothesized that NAT10-mediated ac4C modification directly regulates DYRK1A expression. We performed NAT10-RIP qPCR assay and observed an increased binding between NAT10 protein and CDS domain of DYRK1A RNA in senescent cells (Figure. 5d). To further investigate whether transcriptional regulation of DYRK1A by NAT10 in senescent cells, the DYRK1A promoter was cloned into a luciferase reporter plasmid. No significant change in luciferase activity of the DYRK1A promoter was observed after knocking down NAT10 in senescent cells (Supplementary Figure. 5d).

It has been reported that NAT10 enhances mRNA acetylation, which subsequently increases mRNA stability and translation23,31. We assumed that the increased expression of DYRK1A might be due to post-transcriptional regulation. To validate this, NCM460 cells were treated with the actinomycin D (Act-D). We observed that DYRK1A mRNA stability was increased in senescent cells. However, upon NAT10 knockdown in senescent colon epithelial cells, DYRK1A mRNA stability was reduced (Figure. 5e). Previous studies have shown that ac4C modification within the CDS region can regulate not only mRNA stability but also its translational efficiency. In line with this, polysome profiling followed by qPCR quantification demonstrated a reduced association of DYRK1A mRNA with heavy polysomes upon NAT10 knockdown, supporting that NAT10-mediated ac4C modification also promotes DYRK1A translation (Figure. 5f).

We next confirmed that overexpression of DYRK1A could rescue the senescence reduction effect induced by NAT10 knockdown (Figure. 5g), along with downregulation of IL-1β and IL-6 mRNA expressions (Figure. 5h), γ-H2AX, p21 protein expressions (Figure. 5i), and upregulation of caspase3/7 activity in senescent cells (Figure. 5j).

A significant upregulation of DYRK1A mRNA and protein was observed in replicative senescent colon epithelial CCD841 cells (Supplementary Figure. 6a). Additionally, acRIP-qPCR and RNA decay assays corroborated that the ac4C modification on CDS domain of DYRK1A mRNA was enriched, accompanied by increased mRNA stability in replicative senescent colon epithelial cells (Supplementary Figure. 6b,c). We then knocked down DYRK1A in passage 15 CCD841 cells and observed a marked increase in EdU⁺ proliferating cells (Supplementary Figure. 6e) ,and a reduction in SA-Gal⁺ senescent cells (Supplementary Figure. 6f), further supporting the role of DYRK1A in promoting colonic epithelial senescence. Moreover, overexpression of DYRK1A could rescue the reduction in the senescence phenotype caused by NAT10 knockdown in CCD841 cells (Supplementary Figure. 6g,h).

Targeting NAT10/DYRK1A axis delays colon senescence in mice.

We treated aged mice with the DYRK1A inhibitor Harmine (10 mg/kg, intraperitoneally, every three days for two months) (Supplementary Figure. 7a). Notably, Harmine treatment led to a significant increase in Ki67⁺ epithelial cells in colonic crypts (Supplementary Figure. 7b), suggesting enhanced epithelial regenerative capacity. In addition, Harmine exhibited well tolerability in aged mice, without significant changes in serum biochemistry or histopathology of key organs (Supplementary Figure. 7c,d).

To further determine the functional relevance of the NAT10/DYRK1A axis in regulating colonic aging, we next performed AAV-mediated gene manipulation in aged mice. Knockdown of Nat10 via AAV-shNat10 delivery system significantly alleviated colonic senescence, as evidenced by improved epithelial morphology and reduced expression of p16 and p21. Re-expression of DYRK1A (AAV-Dyrk1a) reversed these protective effects, restoring colonic senescence as comparable to control group (Supplementary Figure. 7e,f).

NAT10 knockdown or pharmacological inhibitor treatment in aged mice promotes intestinal organoid regeneration in vivo

We and others have demonstrated that impaired intestinal epithelial regenerative capacity in intestinal organoids is a key characteristic of cell senescence32-34. We then isolated colonoids from Young-WT and Aged-WT mice, as well as Aged-Nat10+/- mice (Figure. 6a). Results revealed that colonoids derived from Aged-Nat10+/- mice exhibited superior stemness and proliferative capabilities compared to those from Aged-WT mice. Specifically, Aged-Nat10+/- colonoid displayed a higher proportion of budding, increased EdU level (Figure. 6b), faster budding growth rate (Figure. 6c), and elevated expression of Lgr5 protein (Figure. 6d). Additionally, the enhanced proliferation and differentiation capacities of Aged-Nat10+/- colonoid were further validated by the downregulation of p21 and the upregulation of Pcna and Muc2 (Figure. 6e,f). Remodelin was administrated every three days into aged mice for two months and colonoids were isolated (Supplementary Figure. 8a). We found an increased proportion of budding colonoid (Supplementary Figure. 8b) and Lgr5 protein expression (Supplementary Figure. 8c). Enhanced proliferation and differentiation capacities were also observed in the Remodelin treatment group (Supplementary Figure. 8d,e). Taken together, these results indicate that deletion with Nat10 enhances cell stemness and proliferation capacity in colonoid from aged mice.

Previous studies have reported that in colonoids, LPS can increase ER stress, leading to DNA damage and reduced proliferation capacity 35. We validated the appropriate LPS concentration for Aged-WT colonoid in an in vitro model (Supplementary Figure. 8f). After treating with complete culture medium containing 5μg/ml LPS (Figure. 6g), we found that Aged-Nat10+/- mice or Remodelin-treated Aged-WT colonoids maintained relatively higher levels of Lgr5 mRNA (Figure. 6h), and a higher proportion of budding colonoid (Figure. 6i,j). Aged-Nat10+/- or Remodelin-treated Aged-WT colonoids exhibited a reduction of γ-H2AX and Dyrk1a (Figure. 6k).

Targeting the NAT10/DYRK1A axis alleviates colitis in aged mice

Intestinal aging aggravates the susceptibility and severity of ulcerative colitis6,17. We further investigated the impact of intraperitoneal injection of NAT10 inhibitor Remodelin on DSS-induced colitis in Aged-WT mice (Supplementary Figure. 9a). Interestingly, Remodelin treatment markedly reduced body weight loss in aged mice (Supplementary Figure. 9b). Results revealed that a decrease in ac4C modification levels in Aged-WT mice following Remodelin treatment in colon tissues (Supplementary Figure. 9c). The Remodelin-treated group exhibited significantly reduced histopathological damage and the number of γ-H2AX-positive cells in Aged-WT mice (Supplementary Figure. 9d). Additionally, we observed a downregulation of Dyrk1a protein level following Remodelin treatment in colitis mice (Supplementary Figure. 9e).

To confirm the role of NAT10 in aged colitis, we then treated Aged-Nat10-WT or Nat10+/- littermate mice with DSS-containing water for 7 days (Figure. 7a). Compared to Nat10-WT mice, Aged-Nat10+/- mice showed significantly reduced body weight loss (Figure. 7b), colon shortening (Figure. 7c), as well as lower colonoscopy score and histopathological score (Figure. 7d,e). Correspondingly, RNA ac4C levels were decreased in the colons of Aged- Nat10+/- mice (Figure. 7f). Based on these results, we hypothesized that upregulation of Nat10 may exacerbate colitis in aged mice, and depletion with Nat10 protein could alleviate DSS-induced colitis. Results confirmed a relative reduction in the number of γ-H2AX-positive cells, along with significant increase in Ki67-positive cells, goblet cells (Figure. 7g,h). The mRNA levels of p21 and intestinal inflammatory genes (Il-1β, Il-6, and Tnf-α) were significantly reduced in the colonic tissues of Aged-NAT10+/- mice (Figure. 7i). Additionally, Dyrk1a and γ-H2AX protein levels were decreased, while the barrier protein Ocln was upregulated (Figure. 7j,k). These findings further prompted us to investigate the role of Dyrk1a in colitis. We then used Harmine, a well-established Dyrk1a inhibitor, to treat aged mice in colitis model followed by colonoscopic examination (Supplementary Figure. 9f). Enema treatment with Harmine (10mg/kg) for 3 days significantly promoted the healing of colonic mucosal ulcers compared to Vehicle control group (Supplementary Figure. 9g).

To further determine the role of NAT10/DYRK1A axis in elderly-onset colitis, we performed AAV-mediated gene manipulation in aged mice (Figure. 7l). Knockdown of Nat10 via AAV-shNat10 significantly alleviated DSS-induced colitis, as reflected by reduced body weight loss and lower histopathological scores. Re-expression of DYRK1A (AAV-Dyrk1a) reversed these protective effects, restoring colitis severity (Figure. 7m,n). These findings further support that targeting the NAT10/DYRK1A axis play a critical role in mitigating colitis in aged mice.

NAT10 and DYRK1A highly expressed in the aged colon and elderly UC tissues.

Immunohistochemical analysis of colon biopsy samples in our clinical cohort further confirmed the upregulation of NAT10 and DYRK1A in the colonic mucosa of healthy elderly individuals as well as elderly-onset UC patients (Figure. 8a-c). In addition, a positive correlation between NAT10 and DYRK1A immunohistochemical scores was observed in UC biopsy samples (Figure. 8d). Moreover, NAT10 and DYRK1A expression levels were higher in colonic mucosa from elderly UC patients with high endoscopic Mayo scores compared with those with low scores (Figure. 8e). Collectively, these findings suggest that the NAT10/DYRK1A axis may hold translational relevance and represent a potential therapeutic target for elderly-onset colitis.

Methods
Cell culture

The colon epithelial cell line NCM460 (Cellosaurus, #305430, RRID: CVCL_0460) was cultured in RPMI 1640 medium (GIBCO, Waltham, MA, USA), and the finite colon cell line CCD841 (ATCC, CRL-1790, RRID: CVCL_2871) was maintained in Eagle's minimum essential medium (ATCC, Manassas, VA, USA). Each medium was supplemented with 10% fetal bovine serum (ExCell Bio, Suzhou, China) and 1% penicillin-streptomycin (Beyotime, Shanghai, China). All cells were incubated at 37°C in a humidified atmosphere with 5% CO2.

Cell senescence induction

To induce replicative senescence, we performed consecutive passages of CCD841 cells. To induce senescence using Doxorubicin (DOX) (Sigma Aldrich, St. Louis, MO, USA), NCM460 or CCD841 cells were cultured in the presence of DOX (300 nM) in complete culture media for 72 h. Cells were maintained for an additional 5 days after DOX withdrawal before senescence assays were conducted.

For the H₂O₂-induced senescence model (H-Sen), we adapted protocols for colorectal cancer cell lines58, human fibroblasts59 and optimized the conditions accordingly. In brief, NCM460 cells were first exposed to 250 μM H₂O₂ for 48 hours, followed by incubation in medium containing 50 μM H₂O₂ for an additional 6 days to establish a stable senescent phenotype. The H₂O₂-containing medium was replaced daily throughout the induction period.

SA-Gal staining

SA-Gal activity was assessed using a SA-Gal staining kit (Beyotime, Shanghai, China). In brief, cells were rinsed three times with phosphate-buffered saline (PBS) and fixed at room temperature (RT) for 15 minutes with the fixing solution included in the SA-Gal staining kit. After being washed three more times with PBS, the cells were incubated overnight at 37°C in senescence detection solution. The stained cells were subsequently examined under a light microscope.

Cell Cycle Analysis

Cell cycle distribution was analyzed by propidium iodide (PI) staining followed by flow cytometry. Briefly, cells were collected and fixed in 70% ice-cold ethanol at −20°C for at least 2 h. After washing with PBS, cells were incubated with RNase A (100 µg/mL) and PI (50 µg/mL) for 30 min at room temperature in the dark. Samples were analyzed on a flow cytometer and processed using FlowJo software.

Luciferase reporter assay

DYRK1A promoter region ((−2000 to +100 bp) relative to the transcription start site) into the pGL4.10 firefly luciferase vector. The Renilla luciferase plasmid pRL-TK was co-transfected for normalization. The dual-luciferase reporter assay was conducted using the Dual-Luciferase Reporter Assay System (Vazyme, DD1205, China). Cells were plated in 6-well plates and transfected with Lipofectamine 3000. CMV-driven firefly luciferase plasmid was included as a positive control. After 24 hours of transfection, Firefly and Renilla luciferase activities were measured according to the manufacturer’s instructions. Firefly luciferase activities were normalized to Renilla luciferase activities, and the ratio of Firefly to Renilla luminescence was calculated.

EdU assay

EdU incorporation was assessed using the EdU Imaging Kit (APEXBIO, K1076, USA) following the manufacturer’s instructions. Cells were exposed to 10 μM EdU for 1 hour, harvested by trypsinization, and washed with PBS. They were then fixed with 4% paraformaldehyde for 20 minutes and permeabilized in 0.1% Triton X-100 for 20 minutes. After two additional PBS washes, single-cell suspensions were incubated with the EdU detection reagent for 30 minutes at room temperature in the dark. Samples were analyzed on a flow cytometer and processed using FlowJo software.

Quantitative PCR

Total RNA was extracted from cells or tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). A magnetic mRNA isolation kit (NEB, Ipswich, MA, USA) was utilized to isolate poly (A)+ mRNA from total RNA. Quantitative PCR (qPCR) was carried out with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China,) on a Light Cycler 480 II system (Roche, Basel, Switzerland). Reactions were performed in triplicate, and β-actin was used for mRNA normalization. Primer sequences for qPCR are listed in Supplementary Table 2. The 2−ΔΔCt method was employed to determine relative mRNA expression levels.

Liquid chromatography-tandem mass spectrometry for RNA modification dection

A magnetic mRNA isolation kit (NEB, Ipswich, MA, USA) was used to isolate poly (A)+ mRNA from total RNA. The isolated mRNA (200 ng) was digested into nucleosides using 0.5 U nuclease P1 (FUJIFILM Wako, Tokyo, Japan) in 20 μ L of buffer containing 10 mM ammonium acetate, pH 5.3, at 42 °C for 6 hours. Subsequently, 2.5 μL of 0.5 M MES buffer, pH 6.5, and 0.5 μL shrimp alkaline phosphatase (NEB, UTSA) were added, followed by incubation at 37 °C for an additional 6 hours. The mixture was then analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Nucleosides were separated using ultra-performance liquid chromatography on a C18 column and detected by a triple-quadrupole mass spectrometer (AB SCIEX QTRAP 5500) in positive-ion multiple reaction monitoring mode. Mass transitions at m/z were monitored and compared to standard samples (Zz Standard, Shanghai, China), and nucleoside concentrations in the RNA samples were quantified by matching signal intensities to standard curves.

Plasmid and lentivirus construction

Full-length NAT10-WT, NAT10-K290A and DYRK1A was amplified and cloned into a pLVX-puro plasmid. HEK 293 T cells were transfected with above mentioned plasmids and the virus packing plasmids using PolyJet transfection reagent (SignaGen, Rockville, MD, USA). The viral supernatants were harvested and enriched via centrifugation.

SiRNAs construction and transfection

For transient knockdown, siRNAs targeting the NAT10 mRNA and DYRK1A mRNA were purchased from GenePharma (Shanghai, China). siRNA duplexes were transfected using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA)). Transfection was performed according to the manufacturer’s instructions. The siRNA sequences used in this study are listed in Supplementary Table 3.

RNA immunoprecipitation-TqPCR

Cells were lysed using RNA immunoprecipitation (RIP) buffer (25 mM Tris, pH 7.4, 150 mM KCl, 0.5 mM DTT, and 0.5% NP-40) supplemented with RNase inhibitors (1:500, Beyotime, Shanghai, China). NAT10 antibodies (2 μg, Abcam, Cambridge, UK) or the anti-IgG (2 μg, Beyotime, Shanghai, China) were added to the RIP buffer and incubated overnight at 4 °C. Then, Protein A/G magnetic beads were added to the mixture and incubated for 4 h at 4 °C, followed by washing three times with NT2 buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% NP-40). Input and immunoprecipitated RNAs were isolated using TRIzol, followed by qRT-PCR.

Acetylated RIP-seq

Total RNA was extracted using the previously described method and then heated to 70 °C for 6 minutes. The reaction was halted by the addition of EDTA. The fragmented RNA was subsequently purified using the Zymo RNA Clean and Concentrator-25 kit (R1017; Zymo Research, Costa Mesa, CA, USA). The purified RNA was incubated with an anti-ac4C antibody and Dynabeads Protein G (10004D; Invitrogen, Carlsbad, CA, USA)) at 4 °C for 6 hours. Following the manufacturer’s guidelines, the immunoprecipitated RNA was collected. Library construction was performed with the EpiTM Mini LongRNA-seq Kit (E1802; Epibiotek) in accordance with the provided protocol. Library quality was assessed using a Bioptic Qsep100 Analyzer (Bioptic). Sequencing was carried out on the NovaSeq high-throughput platform by Epibiotek (Guangzhou, China).

Acetylated RIP-qPCR assay

The ac4C modifications in individual genes were determined using acetylated (ac)RIP-qPCR assay. Briefly, cells cultured in a 10-cm plate were washed three times with ice-cold PBS and harvested by scraping in 1 mL of RIP buffer supplemented with RNase inhibitors. The TRIzol reagent was used for total RNA isolation. One-tenth of the total RNA was used as the input. Then, 80 μg of total RNA and 2 μg of ac4C antibody were added to RIP buffer supplemented with RNase inhibitors (1:500) and incubated overnight at 4 °C. Then, prewashed Protein A/G magnetic beads were added to the mixture and incubated for 4 h. Following five washes, the acetylated RNA was eluted from the magnetic beads using TRIzol. Eluted RNAs were precipitated using ethanol and dissolved in RNase-free water. The enrichment of certain fragments was detected using qPCR. The proportion of ac4C-enriched RNA to input RNA is presented as the ac4C level.

Western blotting

Total protein was extracted using RIPA lysis buffer supplemented with protease inhibitors. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% bovine serum albumin (BSA) for 2 hours at room temperature (RT), the membranes were incubated overnight at 4°C with the following primary antibodies: NAT10 (1:1000, Abcam, ab194297, UK), p53 (1:1000, 10442-1-AP,Proteintech, Rosemont, IL, USA), p21 (1:1000, 10355-1-AP and 28248-1-AP, Proteintech, USA), p16 (1:1000, Proteintech, 10883-1-AP and 28416-1-AP USA), Occludin (1:1000, Proteintech, 27260-1-AP, USA), Cyclin D1 (1:1000, CST, #2978, USA), γ-H2AX (1:1000, ABclonal Technology, AP0687, MA , USA), DYRK1A(1:1000, Bioss, bs-11834R, China), LMLN(1:1000, Bioss, bs-18180R, China), THAP5 (1:500, OriGene, Rockville, MD, USA), Lgr5 (1:1000, Bioss, bs-20747R, China) and β-actin (loading control) (1:1000, CST, #4967, Danvers, MA, USA). Universal antibody diluent (NCM Biotech, Suzhou, China) was used for antibody dilution. The membranes were then washed three times with Tris-buffered saline containing Tween 20 (TBST) and incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Beyotime, Shanghai, China). Protein signals were detected using the ECL Western Blot Substrate (NCM Biotech, Suzhou, China) and visualized on an Azure 600 Gel Imaging System (1.6.3.1211, Azure Biosystems, Dublin, CA, USA)

RNA ac4C dot blot assay

A total of 2μl containing 1 μg of RNA was heated to 65 °C for 5 minutes and subsequently transferred onto a Hybond-N+ membrane (GE Healthcare, Wauwatosa, WI, USA). The membranes were UV crosslinked for 30 minutes and stained with 0.02% methylene blue (Sangon Biotech, Shanghai, China) to visualize the input RNA. After blocking with 5% BSA, the membranes were incubated overnight at 4°C with an ac4C-specific antibody (diluted 1:250, Abcam, UK). Following three washes with TBST, each lasting 5 minutes, the membranes were treated with horseradish peroxidase-conjugated anti-IgG for 2 hours. The dot blot results were visualized using the Azure 600 Gel Imaging System (Azure Biosystems, Dublin, CA, USA) and quantified with ImageJ software. Methylene blue staining served as the loading control.

RNA decay assay

In brief, actinomycin D (Selleck, Houston, TX, USA) was added to the culture medium at a final concentration of 5 μg/mL. Cells were harvested at 0, 2, and 4 hours. Total RNA was then isolated, and qPCR was conducted to quantify the relative levels of DYRK1A mRNA compared to the 0-hour time point.

RNA sequencing

Total RNA was extracted as described above and assessed with an Agilent Bioanalyzer and NanoDrop. High-quality RNA was used for library construction. RNA-seq libraries were prepared using the Illumina Stranded mRNA Prep Ligation kit (Illumina) and sequenced on an Illumina NovaSeq X Plus platform (Cosmos Wisdom, Hangzhou, China).

Colony formation assay

Cells were seeded into 6-well plates (50,000 cells per well) and cultured in the presence of a senescence inducer, as above described. Afterwards, the cells were transfected and cultured for another 2-4 days. At the end of the assay, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 0.2% crystal violet staining solution (Beyotime, Shanghai, China) for 15 minutes at room temperature (RT).

Immunofluorescence staining

For the cell immunofluorescence assay, cells were cultured on coverslips and fixed for 15 minutes at room temperature (RT) in 4% paraformaldehyde solution. After fixation, the cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 minutes at RT. The coverslips were then washed with PBS and blocked with 3% bovine serum albumin (BSA) in PBS. The primary antibodies were incubated for 2 hours at RT, followed by incubation with the secondary antibody for 1 hour at RT. Immunofluorescence analysis was performed using the γ-H2AX primary antibody (1:100, ABclonal Technology, USA), DYRK1A (1:100, Bioss, China), and Alexa 568-conjugated anti-rabbit secondary antibody (1:200, Invitrogen, USA).

For the tissue immunofluorescence assay, paraffin-embedded colon tissues were deparaffinized, rehydrated, and subjected to antigen retrieval. The sections were then blocked with 3% bovine serum albumin (BSA) and incubated with the following primary antibodies: anti-Muc2 (Proteintech, 27675-1-AP, 1:500), anti-Ki67 (Servicebio, GB111402, 1:500), anti-Occludin (Servicebio, GB111401, 1:500). After primary antibody incubation, the sections were incubated with the appropriate secondary antibodies.

Tissue HE staining and IHC analysis

Colon tissues were fixed in 4% paraformaldehyde. Hematoxylin and Eosin (HE) staining and Periodic Acid Schiff (PAS) staining were performed to evaluate morphological changes.

For IHC analysis, we collected formalin-fixed, paraffin-embedded biopsy specimens from 22 healthy donors and 37 patients diagnosed with ulcerative colitis at the Second Affiliated Hospital of Zhejiang University School of Medicine. After deparaffinization, rehydration, antigen retrieval, and blocking of non-specific binding, the sections were incubated overnight at 4°C with NAT10 antibody (Abcam, 1:200) or DYRK1A antibody (Bioss, 1:200). The next day, the sections were treated with secondary antibodies for 50 minutes at room temperature. Following this, the sections were stained with DAB, counterstained with hematoxylin, and observed under a Nikon microscope. The proportion of positive cells was quantified and categorized into four groups: 0 (0–5%), 1 (6%–50%), 2 (51%–75%), and 3 (76%–100%). Staining intensity was graded as follows: 0 for no staining, 1 for weak, 2 for moderate, and 3 for strong staining. The final staining score was obtained by multiplying the positive cell percentage by the staining intensity. Information of human samples is provided in the Supplementary Table 4.

DSS-induced colitis model

Acute colitis was induced using DSS (160 110, MP Biomedicals). Two weeks prior to induction, the mice were randomly assigned to different cages for co-housing. They were then administered a 7-day treatment with 3% DSS via oral gavage. All the mice used in this study were housed in the specific pathogen-free facility of the Laboratory. Experimental procedures were conducted in accordance with the guidelines of the Institutional Animal Use.

For colonoscopy in mice, the animals were fasted for 24 hours and anesthetized with isoflurane. An ENDOQ small animal endoscope (ENDOQ-E1, Siboyu Biotechnology Corporation) was used to perform the colonoscopy.

Nat10+/- mice and mice experiments

Nat10-knockout mice were generated and obtained from Shanghai Model Organisms Center, Inc. Briefly, the Nat10+/- mice used in this study were created by deleting exon 4 using a CRISPR/Cas9-based genome editing approach. Genotyping was performed by PCR with primers flanking the deleted region.

All animal studies were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Zhejiang University. All mice used in this study were on a C57BL/6 genetic background and maintained at a specific pathogen-free (SPF) level animal facility in Animal Experiment Center, Zhejiang Chinese Medical University. The ambient temperature was maintained at 20–22°C with a relative humidity of 45–65%. Mice were provided a standard laboratory diet, water and were kept in ventilated cages under a 12-hour light/12-hour dark cycle.

NAT10 and DYRK1A inhibitor treatment

Remodelin was prepared in PEG300 and TW-20 and administered intraperitoneally at a dose of 20 mg/kg. Harmine was prepared in PEG300 and TW-20 and administered intraperitoneally at a dose of 10 mg/kg. A solvent with the same composition and volume was used as the vehicle group.

For mouse enema treatment, the mice were fasted for 24 hours and anesthetized with isoflurane. A polyethylene catheter was inserted into the colon, approximately 4 cm from the anus, and Harmine or vehicle was slowly administered using a syringe. Following the injection, the mice were kept in an inverted vertical position for at least 1 minute. After recovery, the mice were allowed to resume normal water and food intake.

Nat10 knockdown and Dyrk1a expression in mice

All adeno-associated viruses serotype 8 (AAV8) were purchased from Genechem (Shanghai, China). AAV8-shNat10 and AAV8-shNC (CV762 vector with villin promoter) were used to knock down Nat10 expression in vivo. The shRNA targeting sequence for mouse Nat10 was 5ʹ-GAGCATCTGGATTATGAGATT-3ʹ. Each mouse received 5.0E+11 v.g of virus via tail-vein injection. Dyrk1a overexpression was achieved using adeno-associated viruses (AAV8-Dyrk1a and AAV8-Vector, The GV411 vector with CMV promoter). Each mouse received 1.0E+12 v.g of virus via tail-vein injection.

Colonoid construction and culture

Colon tissues from C57BL/6 mice were dissected longitudinally and rinsed three times with cold Dulbecco's Phosphate-Buffered Saline (DPBS) containing penicillin–streptomycin to clear out contents and mucus. The tissues were then cut into small pieces and incubated in 5 mM EDTA on a rotor at 4°C for 40 minutes. Crypts were released by mechanical shearing with pipette tips in PBS with 0.01% BSA. After passing through a 100 μm cell strainer, 200 crypts were combined with 50 μl of Matrigel (Corning #356231) and plated as a dome in the center of a pre-warmed 24-well plate. The plate was incubated at 37°C with 5% CO2 for 10 minutes to solidify. Complete Intesticult medium (Stemcell Technologies #06005) was added to support colonoid growth and replaced every 3 days. Passaging was performed after 7 days of culture.

For the in vitro lipopolysaccharide (LPS) treatment experiment, LPS (Sigma-Aldrich, L4391, USA) was added to the complete culture medium at a concentration of 5 μg/ml for colonoid cultivation. On day 3 of the culture period, the medium was replaced with LPS-containing medium. For the NAT10 inhibitor experiment, the NAT10 inhibitor Remodelin (10 μM) or vehicle was added to the complete colonoid medium. Colonoids were photographed daily starting from day 7 of the culture period using an inverted microscope to evaluate regenerative growth.

Statistics and reproducibility

Each experiment was performed with at least three biological replicates. Representative images for fluorescence staining, PAS staining, H&E staining and SA-Gal staining are shown, each of these experiments included at least three independent samples and was repeated at least three times. Statistical analyses were performed using GraphPad Prism 10.0 Software (GraphPad, Inc., San Diego, CA, USA). Data were expressed as mean ± SD (standard deviation). Differences between groups were analyzed by Two-tailed unpaired Student’s t-test and two-way ANOVA with Tukey’s multiple comparisons. Correlations were performed using the Spearman correlation test. A p-value ＜0.05 was regarded as statistically significant. No statistical method was used to predetermine the sample size. The sample sizes (“n”) and the statistical tests are described in each figure legend, exact p values are provided in the Source Data (*p < 0.05, **p <0.01, and ***p <0.001).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, Chen and colleagues investigated the role of N-acetyltransferase 10 (NAT10)-mediated mRNA N4-acetylcytidine (ac4C) modification in colonic epithelial senescence and elderly-onset colitis. They demonstrated that NAT10 stabilizes DYRK1A mRNA via ac4C modification and thereby promotes epithelial senescence. The involvement of NAT10 in aging has been reported, and the concept is not very novel. Technically, the acRIP-seq needs more validations, and the in vivo data is not convincing. In addition, there are controversies about whether Remodelin is a specific inhibitor for NAT10, and the authors should be very cautious on the conclusions based on in vivo data generated by Remodelin.\nHere are the detailed comments:\n1. The in vivo data is not solid and convincing. As shown in Figure 3e, Aged-Nat10+/- mice exhibit extremely low or even non-existent levels of Nat10, which is much lower than that of Young-WT mice from immunostaining results. Does the effect of heterozygotes have such a significant impact? Does the alleviation of aging manifested by Aged-Nat10+/- mice occur systemically? Using heterogeneous mice for in vivo experiments is not convincing, the authors should use colon-specific Cre and use homogenous KO mice for these in vivo studies.\n2. The acRIP-seq data is very limited. How many peaks has been identified for each sample? and the number of genes corresponding to the peaks. Such information about acRIP-seq should be mentioned in the manuscript. Particularly, in Figure 4d and e, up ac4c peaks between H-Sen vs Control and down ac4c peaks between H-Sen NAT10-si vs NC-Si, the two parts should correspond to a series of peaks and genes regulated by NAT10 during the aging process, but the intersection is only 4 genes. Why are there so few common genes between them? More details should be shown. Among the four genes, THAP5 shows the largest fold change among the three samples. Why did acRIP-qPCR fail to detect it? In addition, please provide the IGV visualization of the acRIP-seq data of control and NAT10i groups showing ac4C peaks on DYRK1A and other candidates.\n3. As a targeted therapeutic approach for elderly-onset UC, there is still a lack of toxicity data, such as the systemic effects of NAT10 inhibition. Given that the NAT10/DYRK1A axis plays such a significant role in colon senescence, does it also exert its effects in other cells or tissues? Did Remodelin-treated aged mice exhibit such phenotypes as p53 decrease and cell proliferation in other tissues?\n4. Global impact on mRNA stability is lacking. What are the global changes in mRNA half-life after NAT10 knockdown under the senescent cell context? Was any transcriptome-wide RNA stability analysis (e.g., Actinomycin D chase followed by RNA-seq) performed to examine changes in mRNA half-life following NAT10 knockdown? This analysis would help clarify whether NAT10 broadly regulates RNA stability beyond its effect on DYRK1A.\n5. Effect on DYRK1A translation is lacking. In the mechanistic study (line 205–206), the authors mentioned that “We hypothesized that NAT10-mediated ac4C modification directly regulates DYRK1A expression.” However, it is unclear whether translation efficiency is also affected. Assessing DYRK1A translation (e.g., via polysome profiling or ribosome footprinting) would help complete the mechanistic picture.\n6. While NAT10 is proposed as a therapeutic target, patient sample validation is lacking. Including clinical data would strengthen the study’s translational value.\n7. Do Remodelin and Harmine have similar effects in delay of colon senescence in aged mice? Which one is better?\n8. In Figure 4b, which sample's ac4C distribution is being shown here? Corresponding to Figure 4c, the distribution of all three samples should be included.\n9. In Figure 6b and 7k, Colonoids should be marked with a scale, and figure legends require specific scale bars.\n10. Lack of the letter l in Figure 7’s figure legends.\n11. Some methodological descriptions lack detail, such as the specific parameters for RNA ac4C modification assays. Some statistical methods are not clearly described, such as whether appropriate corrections were applied for multiple comparisons. Clarifying statistical procedures is highly recommended.\n12. Need grammar and language refinement: Some sentences would benefit from grammatical corrections and clearer phrasing. Examples include but not limited: Line 47: \"self-renew\" should be \"self-renewal\"; Line 56: \"inhibitors treatment with NAT10 or DYRK1A\" should be \"treatment with NAT10 or DYRK1A inhibitors\"; Line 58: \"rejuvenate intestinal aging\" would be clearer as \"rejuvenate the aged intestine\" or \"reverse intestinal aging\"; Line 148: \"a RNA ac4C-dependent manner\" should be \"an RNA ac4C-dependent manner”.", "reviewer_2": "The authors of the submitted manuscript demonstrated that the NAT10/DYRK1A axis regulates the response during the late(old) onset of colitis in mice. In vitro and in vivo experiments showed that the levels of NAT10 regulate colonic senescence status, and the NAT10 stabilizes DYRK1A mRNA. In summary, it is an interesting concept, and the authors provided plenty of data to support their hypothesis. I have several comments that I would like the authors to address.\nFigure 3e. IF, Occludin, please include the colonic crypts that show the entire crypt. Due to the angle of the tissues, we do not see the full crypt (specifically the crypt included in the center of the image).\nFigure 3e. IF LGR5, I am unsure whether the author noticed that the stem cell marker staining is in multiple crypt sections while it should be localized to the bottom. As such, this image is misleading; not everybody works for Western, IF, etc. The authors should include in situ hybridization instead.\nThe authors delivered Remodelin via IP; however, it is an orally available drug. Please explain.\nFigure 7 and extended Figure 7: please provide weight information throughout the experiments with clinical and histological scores.\nNotably, the authors show that aged-Nat10+/- mice have increased levels of Ki67 (Figure 3e), which suggests that they will be more susceptible to the injury (as proliferative cells are more susceptible to the injury); however, the results are the opposite. Please explain.\nFigure 7k: Please include IHC stains from tissues of healthy young and aged patients (non-IBD).\nPlease follow the nomenclature of murine and human genes and proteins and proofread your manuscript. There are multiple minor grammatical and orthographical mistakes.", "reviewer_4": "Using intestinal models, both in vitro and in vivo, this study investigates the role of NAT10 in senescence. Their data suggest that NAT10 is upregulated during senescence, contributing to the phenotype through asC4 modification of DYRK1A mRNA in culture. Interestingly, the NAT10/DYRK1A axis is also upregulated in UC models in humans and mice. In Nat10+/- mice, Nat10 levels are substantially reduced: Using this model, they show the critical role of this axis in the colitis of aged mice. The age-associated alteration in the NAT10/DYRK1A axis is potentially interesting, but the link to senescence is weak.\nFig. 1: Overall, the senescence assay is not robust. Are these cell models well-established (no references are provided)? With a quick search, I was not able to find previous studies characterizing senescence in these cell lines. Sa-b-gal is a good marker, but it is not necessary or sufficient for defining senescence. Validation of persistent cell cycle arrest is essential. In addition, it would be helpful if they added blots of p16, a more specific senescence marker (Fig. 1e).\nIn the doxycycline model, they treated the cells for 72 h. I assume they conducted senescence assays at this point. If so, it may be too early. Typically, cells are maintained for additional days without drugs to fully establish senescence. Of course, this can vary depending on the cell type, which is why a comprehensive characterization of senescence is essential before correlating it with other phenotypes, such as an mRNA modification. It would also be helpful if the authors could briefly describe the H2O2 model.\nThese senescence assays are all under the assumption that these cells are resistant to cell death. Or are we looking at residual cells surviving the drug treatment?\nFig. 2 suggests that the knockdown of NAT10 promotes apoptosis rather than bypassing senescence during dox treatment, even though overall p53 signaling and DDR are alleviated. I wonder what could be a mechanism for this? Is the cell cycle maintained (western for cell cycle factors, BrdU/EdU assays would be helpful)?\nSenescence is generally associated with 'persistent' DDR, which is rather modest. However, Fig. 2c suggests a highly robust DDR in these cells. this is perhaps consistent with my earlier comment on the timing of Dox-senescence (72h might be too early). Is it possible that the phenotype is associated with DDR rather than senescence?\nFig. 3: How about cell death? Do the authors observe enhanced cell death in aged Nat10+/- intestine? This might be more pronounced in the colonoid experiments, particularly in the presence of LPS (Fig. 6)?\nFig. 4 (acRIP-seq): I couldn't find information on the number of biological replicates. How consistent are the replicates? Also, it would be helpful if they provided more general information, such as the total number of peaks identified in each condition, particularly considering that only a small number of differential peaks were identified (Fig. 4e).\nFig. 5e: No details on the reporter construct are provided. Also, it lacks positive controls.\nFig. 7f: The authors tested SASP genes here. Do they mean that these cytokines are expressed by 'senescent' epithelial cells? Or are they expressed by immune cells? Are these SASP genes also upregulated during ageing with no DSS (e.g., Fig. 3, 6)?\nRNAi experiments: it is highly encouraged to test multiple target sequences to alleviate off-target effects.\nNat10 knockout mice: It would be helpful if the authors could provide more details, such as the targeting strategy, in the Methods section.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Re: Targeting NAT10 Alleviates Colonic Senescence and Elderly-Onset Colitis by Disrupting RNA Acetylation-Mediated DYRK1A Stabilization\nNature Communications Reference: NCOMMS-25-11798\nReviewers' Comments:\nReviewer #1 (Remarks to the Author):\nIn this study, Chen and colleagues investigated the role of N-acetyltransferase 10 (NAT10)-mediated mRNA N4-acetylcytidine (ac4C) modification in colonic epithelial senescence and elderly-onset colitis. They demonstrated that NAT10 stabilizes DYRK1A mRNA via ac4C modification and thereby promotes epithelial senescence. The involvement of NAT10 in aging has been reported, and the concept is not very novel. Technically, the acRIP-seq needs more validations, and the in vivo data is not convincing. In addition, there are controversies about whether Remodelin is a specific inhibitor for NAT10, and the authors should be very cautious on the conclusions based on in vivo data generated by Remodelin.\nResponse: We sincerely thank you for the constructive comments. We have conducted additional experiments and the major revisions as below:\n1. To substantiate in vivo findings, we employed AAV-mediated gene modulation in aged mice. Administration of AAV-shNat10 significantly alleviated colon senescence and DSS-induced colitis, whereas re-expression of DYRK1A reversed these phenotypes. In parallel, pharmacological inhibition of either NAT10 or DYRK1A was well tolerated in aged mice and similarly ameliorated colonic senescence. Together, these additional data consistently demonstrate that targeting the NAT10/DYRK1A axis mitigated colon epithelial aging.\n2. For mechanistic validation, Actinomycin D chase RNA-seq and polysome profiling were performed to further evaluate the effects of NAT10 knockdown on DYRK1A mRNA stability and translation. These results support that DYRK1A is a key downstream target regulated by NAT10 in the context of colonic senescence. We updated the acRIP-seq dataset and have provided a more detailed description in the revised manuscript.\n3. The relationship between NAT10 and DYRK1A expression and disease severity was evaluated in elderly UC patients, with patients stratified by endoscopic Mayo scores. Moreover, the expression levels of NAT10 and DYRK1A were further examined in colonic mucosal biopsy samples from healthy young and aged human donors.\nWhile previous studies have suggested that NAT10 may alleviate aging phenotypes in progeroid models (Revised discussion section Line 358-360), these syndromes are typically caused by specific genetic mutations and differ from physiological aging. The role of NAT10 in organ-specific context such as the colon, remains largely unexplored. Our findings, for the first time, suggest that NAT10 serves as an epigenetic regulator in maintaining colonic epithelial homeostasis during aging. The specificity of Remodelin as a NAT10 inhibitor remains under debate. Nevertheless, Remodelin continues to be widely used in recent research as a tool compound to investigate NAT10‑associated biological processes in both cellular and animal models1,2. In our study, Remodelin was employed as a complementary pharmacological approach to support in vivo findings.\n1. The in vivo data is not solid and convincing. As shown in Figure 3e, Aged-Nat10+/- mice exhibit extremely low or even non-existent levels of Nat10, which is much lower than that of Young-WT mice from immunostaining results. Does the effect of heterozygotes have such a significant impact? Does the alleviation of aging manifested by Aged-Nat10+/- mice occur systemically? Using heterogeneous mice for in vivo experiments is not convincing, the authors should use colon-specific Cre and use homogenous KO mice for these in vivo studies.\nWe fully agree that using colon-specific Cre and homozygous knockout models would provide a more definitive approach to dissect the function of NAT10 in the colon epithelium. During our early attempts to generate Nat10 homozygous knockout mice, we observed embryonic lethality associated with complete deletion of the gene. We found that NAT10 expression was markedly reduced in the colonic crypts of Nat10+/−mice (Response Fig.1a), which prompted us to utilize this model for further studies. Nat10+/− mice did not exhibit a significant alteration in overall lifespan under physiological conditions (Response Fig.1b). Nat10+/− mice displayed marked attenuation of colonic epithelial senescence phenotypes, such as enhanced crypt regeneration capacity and reduction in canonical senescence markers, suggesting that partial NAT10 deficiency may contribute to the preservation of colonic epithelial homeostasis during aging. Importantly, the Nat10+/− mice exhibited phenotypes similar to those observed upon pharmacological inhibition of NAT10 using Remodelin, supporting the validity of this approach. These observations were also in line with recent studies demonstrating that NAT10 plays a role in disease models such as synovial aggression, where both heterozygous knockout and Remodelin treatment produced comparable effects3.\nTo further validate these findings, we constructed adeno-associated virus to knock down Nat10 expression in aged mice. Two months after AAV-shNat10 administration, we observed decreased expression of the senescence markers p16 and p21 (Response Fig.1c), along with increased Ki67⁺ proliferative cells and elevated expression of Ocln (Response Fig.1d). These data and methodological details have been incorporated into the revised manuscript (updated Fig. 3f,g).\n120\n100\n80\n60\n40\n20\n0\n0 6 12 18 24 30\nResponse Fig.1 Knockdown Nat10 alleviates intestinal aging in mice.\n)%(lavivruS\na\nYoung-WT Aged-WT Aged-Nat10+/-\nb Aged-\nWT\nAged-Nat10+/-\nWT\nNAT10+/-\nIPAD/01taN\nLog-rankP>0.05\nc d AAV-NC AAV-shNat10\nAAV-NC AAV-shNat10\n#1 #2 #3 #4 #5 #1 #2 #3 #4 #5kDa\np21 25\np16\nβ-actin\nnlcO/76iK\nMonths\na Immunofluorescence analysis of Nat10 expression in Young-WT, Aged-WT, and Aged-Nat10+/- mice. Scale bar, 50 μm.\nb, Gross anatomical morphology and survival curves of Aged-WT (n = 10) and Aged-Nat10+/− mice (n=12 each group).\nc, Western blot analysis of Nat10, p21, and p16 in colon tissues from Aged-WT mice after two months tail-vein injection of AAV-NC or AAV-shNat10.\nd, HE staining and immunofluorescence for Ocln and Ki67 in colon tissues from mice, two months after tail-vein injection of AAV-NC or AAV-shNat10. Scale bar, 100 μm.\nAAV, adeno-associated virus.\n2. The acRIP-seq data is very limited. How many peaks has been identified for each sample? and the number of genes corresponding to the peaks. Such information about acRIP-seq should be mentioned in the manuscript. Particularly, in Figure 4d and e, up ac4c peaks between H-Sen vs Control and down ac4c peaks between H-Sen NAT10-si vs NC-Si, the two parts should correspond to a series of peaks and genes regulated by NAT10 during the aging process, but the intersection is only 4 genes. Why are there so few common genes between them? More details should be shown. Among the four genes, THAP5 shows the largest fold change among the three samples. Why did acRIP-qPCR fail to detect it? In addition, please provide the IGV visualization of the acRIP-seq data of control and NAT10i groups showing ac4C peaks on DYRK1A and other candidates.\nThank you for the valuable suggestions and apologize for any confusion. We performed acRIP-seq with three biological replicates each group. The average changes across these replicates were used for integrated analysis, serving as the basis for the identification of downstream candidate genes (presented in Response Table1). Besides, detailed descriptions of the dataset and analyses were provided in the revised manuscript. Raw sequencing reads were deposited to the SRA database, with the accession number PRJNA1347399.\nSamples Peak number Gene count\nControl-1 3771 3039\nControl-2 8573 5159\nControl-3 3590 2728\nH-Sen-1 5357 3378\nH-Sen-2 4897 3195\nH-Sen-3 3339 2398\nH-Sen-NAT10-Si1 9015 5012\nH-Sen-NAT10-Si2 5496 3497\nH-Sen-NAT10-Si3 3720 2791\nResponse Table1. Summary of ac4C peak numbers and ac4C-enriched gene counts for each acRIP-seq sample.\nFrom the acRIP-seq analysis, 1,228 hyper-acetylated peaks (log₂FC>0) corresponding to 858 genes were identified in the H-Sen group compared with the control group (supplementary dataset1). In contrast, 640 hypoacetylated peaks (log₂FC<0) corresponding to 529 genes were detected in the H-Sen NAT10-Si group compared with the H-Sen group (supplementary dataset2). The corresponding information updated in the Revised results section (Line 193-196).\nTo pinpoint key transcripts regulated by NAT10, we applied filtering criteria. We first identified 32 protein-coding transcripts that exhibited increased ac4C abundance (log₂FC>1.5) in senescent cells (H-Sen) compared to control. Upon NAT10 knockdown in senescent cells (H-Sen-Si), 11 protein-coding transcripts showed reduced ac4C abundance (log₂FC>1.5). The intersection of these datasets yielded four candidate target genes: THAP5, DYRK1A, LMLN, and MVB12A (Updated Fig.4d). We merged the three biological replicates for each acRIP-seq group and performed IGV visualization, as shown in Updated Fig.4h. The IGV visualizations for the other three candidate genes are shown in Updated Extended Data Fig.5d.\nAlthough THAP5 exhibited the prominent change of ac4C level in acRIP-seq data and has been previously reported as a cell cycle inhibitor4, we found that its RNA expression was very low (Ct value>30) in NCM460 cells based on qPCR analysis, which may partly explain the unsuccessful detection in our acRIP-qPCR validation. In addition, we examined the protein levels of THAP5 following NAT10 knockdown and found that silencing NAT10 did not alter THAP5 protein expression (Updated Fig.4i).\nCollectively, through integrative validation at the transcriptomic, epitranscriptomic, and protein levels, our findings support that DYRK1A is the key downstream gene\nFig4\na b\nacRIP-seq schematic Control+NC-Si H-Sen+NC-Si H-Sen+NAT10-Si\nc d\nH-Sen vs Control NAT10 H -S -S i v e s n NC-Si H-Sen vs Control NAT10 H -S -S i e vs n NC-Si\nupac4C peaks-GO downac4C peaks-GO\nHyperacetylated Hypoacetylated\ntranscripts 4 transcripts\n28 7\nac4C peak |log2FC| >1.5\nGene H-Se L n o v g s 2 F C C ontrol NAT1 L 0 H o -S - g S i 2 e v F s n C NC-Si Lo P c e a a ti k on\nTHAP5 2.82 -3.07 5'UTR\nDYRK1A 2.44 -1.86 CDS\nLMLN 1.75 -1.85 3'UTR\nMVB12A 1.53 -1.57 CDS\ne f g\nacRIP-qPCR RNA-seq following ActD\n*** *** *** ***\n** ns ** **\nns ns ** ns\n***\nh DYRK1A(Merged IGV plots） i Control+N\nH\nC\n-S\n-S\nen\ni +N\nH\nC\n-S\n-S\ne\ni\nn+NAT10-Si\nkDa\nTHAP5 40\nDYRK1A 100\nControl+NC-Si\nLMLN 75\nH-Sen+NC-Si\nβ-actin 40\nH-Sen+NAT10-Si\nac4C\nMB\nUpdated Fig.4 DYRK1A as a target of mRNA ac4C modification in regulating\ncolon epithelial cell senescence\na, Schematic illustration of acRIP-seq in NCM460 cells.\nCreated in BioRender. Chen, J. (20 26) https://BioRender.com/j92d013\nb, Pie charts showed the distribution of ac4C peak in the acetylated transcripts in the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups.\nc, GO-BP enrichment analysis of hyperacetylated genes in H-Sen NCM460 cells compared to control NCM460 cells (left). GO-BP enrichment analysis of hypoacetylated genes in H-Sen NCM460 cells with NAT10-Si compared to H-Sen NCM460 cells with NC-Si (right).\nd, Overlapping the potential downstream targets of NAT10-meditated RNA ac4C modification in hyperacetylated gene (H-Sen NC-Si vs Control NC-Si) and hypoacetylated gene (H-Sen NAT10-Si vs H-Sen NC-Si) by the acRIP-seq analysis.\ne, acRIP-qPCR validation of ac4C level alterations in four potential downstream targets across the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups.\nf, qPCR was used to validate DYRK1A and LMLN RNA expression across the three groups. g, Actinomycin D (ActD) chase RNA-seq showing DYRK1A mRNA level change in H-Sen NC-Si and H-Sen NAT10-Si cells.\nh, IGV plots showed that ac4C peaks changes in the CDS domains of DYRK1A mRNA in the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups from acRIP-seq data.\ni, RNA ac4C modifications and THAP5, DYRK1A and LMLN protein levels were detected by dot blot or western blot across the three groups. Data are represented as mean ± SD with individual data points shown (n = 3). Two-way ANOVA analysis with Tukey’s multiple comparison was used for (g). Student’s t-test was used for other analyses. *** p<0.001, ** p<0.01, ns p>0.05.\n3.As a targeted therapeutic approach for elderly-onset UC, there is still a lack of toxicity data, such as the systemic effects of NAT10 inhibition. Given that the NAT10/DYRK1A axis plays such a significant role in colon senescence, does it also exert its effects in other cells or tissues? Did Remodelin-treated aged mice exhibit such phenotypes as p53 decrease and cell proliferation in other tissues?\nThank you for this point. To assess potential systemic toxicity, we conducted additional evaluations on the safety of Remodelin in aged mice. Based on previously reported protocols for intraperitoneal administration of these compounds in mice, aged animals were injected intraperitoneally every three days with Remodelin (20 mg/kg). We then assessed serum biochemistry parameters (Response Fig.2a) and performed histopathological analyses of major organs (including the liver, kidney, lung, and spleen) (Response Fig.2b). The results showed no obvious toxicity or tissue damage, indicating safety profiles for our experimental conditions and these data and methodological details have been incorporated into the revised manuscript (updated Fig. 4e,f).\nTo further explore whether NAT10 inhibition affects senescence and regeneration in other tissues (Response Fig.2c), we performed immunohistochemical staining for p21 and Ki67 in the heart, liver, lung, and kidney of Remodelin-treated aged mice (Response Fig.2d). In the liver, we detected a reduction in p21-positive cells alongside an increase in Ki67-positive staining, indicating reduced senescence and enhanced proliferative capacity. Representative images of liver sections from vehicle- and Remodelin-treated groups are shown in Response Fig. 2e. No significant changes were observed in other detected organs. We then performed in vitro senescence assays using a hepatic cell line (Huh7) and a pulmonary epithelial cell line (A549). NAT10 knockdown or inhibition partially alleviated senescence features in Huh7 cells, whereas no significant effects were observed in A549 cells (Response Fig.2f), suggesting organ- and cell-type specificity in NAT10's anti-aging effects.\nResponse Fig.2 Remodelin delays colon senescence in mice.\na. Serum biochemical parameters (ALT, AST, BUN, CR, LDH, and CK-MB) in aged mice treated with Rem or vehicle. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CR, creatinine; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB isoenzyme.\nb, HE staining of the heart, liver, spleen, lung, and kidney from aged mice treated with Rem or vehicle. Scale bar, 100 μm.\nc. Western blot analysis of Nat10 in the colon of 2, 6,12, and 18-mon aged mice.\nd. IHC staining of Ki67 and p21 in the heart, liver, spleen, lung, and kidney from aged mice treated with Rem or vehicle. Ki67+ and p21+ cells were quantified from 15 random fields for comparison.\ne. Gross morphology of liver from aged mice treated with Rem or vehicle.\nelciheV\nmeR\nc\ne f\nLiver\n7huH\nNC-Si NAT10-Si1 NAT10-Si2\n945A\n100\n80\n60\n40\n20\n)%(llec\n+laG-AS\n120\n90\n60\n30\n)%(llec\n+laG-AS\nHeart Liver Lung Kidney\nYoung Aged Young Aged Young Aged Young Aged\n#1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3 #1 #2 #3\nNat10\nβ-actin\nHuh7 A549\nnsns\n**\n**\nelciheV\nmeR\nHeart Liver Lung Kidney\n76iK:CHI\n12P:CHI\nelciheV\nmeR\nHeart Liver Lung Kidney\n15\n12\n9\n6\n3\ndleiF/sllec\n+76iK\n15\n12\n9\n6 3\ndleif/sllec\n+12P\n25\n20\n15\n10 5\ndleif/sllec\n+12P\n15\n12\n9\n6 3\ndleif/sllec\n+12P\n15\n12\n9\n6 3\ndleif/sllec\n+12P\nns\n**\nns ns\nns\n** ns ns\nelciheV\nmeR\na\nns\nns ns\nns\nns ns\nb\nHeart Liver Spleen Lung Kidney\nd\nf. Analysis of SA-Gal activity in a hepatic cell line (Huh7) and a pulmonary epithelial cell line (A549) after transfected with NAT10-SiRNAs and treated with H O . Scale\n2 2\nbar, 100 μm. Data are presented as mean values ± S.D. Comparisons were performed by two-tailed t test. *** p<0.001, ** p<0.01, ns p>0.05.\n4. Global impact on mRNA stability is lacking. What are the global changes in mRNA half-life after NAT10 knockdown under the senescent cell context? Was any transcriptome-wide RNA stability analysis (e.g., Actinomycin D chase followed by RNA-seq) performed to examine changes in mRNA half-life following NAT10 knockdown? This analysis would help clarify whether NAT10 broadly regulates RNA stability beyond its effect on DYRK1A.\nTo further evaluate the global impact of NAT10 on mRNA stability, we first examined the decay of GAPDH and β-actin mRNAs, which did not exhibit detectable ac4C peaks in our acRIP-seq dataset. Upon NAT10 knockdown, no significant changes were observed in the half-lives of these transcripts, suggesting that NAT10’s effect on RNA stability is selective and more likely dependent on ac4C modification, at least in the context of senescent colonic epithelial cells (Response Fig.3a).\nAs suggested, we performed Actinomycin D chase experiments followed by RNA-seq in senescent colonic epithelial cells with or without knockdown of NAT10. Cells were harvested at 0, 2, and 4 hours after Actinomycin D treatment (Response Fig.3b). To investigate the temporal expression of candidate genes, we compared Log₂(FPKM+1) values at three different time points. Among the four candidates analyzed, DYRK1A exhibited a marked and consistent decrease in mRNA levels over time, whereas the other three genes did not (Response Fig.3c, Updated Fig.4g and Extended Data Fig.5c).\nIn addition, we identified 627 transcripts that were downregulated by more than 1.5-fold in the H-Sen NAT10-Si group compared with the H-Sen NC-Si group at 0 h after Actinomycin D treatment. Notably, the Log₂(FPKM+1) values of these transcripts continued to decrease over time in the NAT10 knockdown group. The detailed information of these transcripts is provided in the supplementary dataset3.\na GAPDH β-actin b\nns\nns\n1.2\n1.0\n0.8\n0.6\n0h 2h 4h\nResponse Fig.3 mRNAs half-life following NAT10 knockdown.\na, GAPDH and β-actin mRNA stability was measured by adding actinomycin D in control, H-Sen, D-Sen NCM460 cells and in H-Sen NCM460 cells with NAT10 knockdown.\nb, The schematic diagram of the experimental procedure for RNA-seq after Actinomycin D treatment in H-Sen NC-Si and H-Sen NAT10-Si cells.\nc, Actinomycin D (ActD) chase RNA-seq showing DYRK1A, THAP5, LMLN and MVB12A mRNA level change in H-Sen NC-Si and H-Sen NAT10-Si cells. Data are presented as mean values ± S.D. Comparisons were performed by two-tailed t test. *** p<0.001, ns p>0.05.\n5. Effect on DYRK1A translation is lacking. In the mechanistic study (line 205–206), the authors mentioned that “We hypothesized that NAT10-mediated ac4C modification directly regulates DYRK1A expression.” However, it is unclear whether translation efficiency is also affected. Assessing DYRK1A translation (e.g., via polysome profiling or ribosome footprinting) would help complete the mechanistic picture.\nWe appreciate this valuable suggestion. In this study, we first observed that DYRK1A mRNA expression was significantly downregulated upon NAT10 knockdown in\n)1+MKPF2gol(\nlevel\nANRm\nevitaleR\nDYRK1A THAP5 MVB12A 1.2 H-Sen+NC-Si\nH-Sen+NAT10-Si\n1.0\nns\n0.8\nns\n0.6 ***\n0h 2h 4h\n)1+MKPF2gol(\nlevel\nANRm\nevitaleR\nH-Sen+NC-Si 1.2\nH-Sen+NAT10-Si\n1.0\n0.8\n0.6\n0h 2h 4h\n)1+MKPF2gol(\nlevel\nANRm\nevitaleR\nH-Sen+NC-Si 1.2\nH-Sen+NAT10-Si\n1.0\n0.8\n0.6\n0h 2h 4h\n)1+MKPF2gol(\nlevel\nANRm\nevitaleR\nc\nLMLN H-Sen+NC-Si\nH-Sen+NAT10-Si\nns\nsenescent colonic epithelial cells. Further analysis revealed that NAT10 modulates DYRK1A expression by regulating the mRNA stability. Previous studies have reported that elevated ac4C modification promotes both mRNA stability and translational efficiency3,5. Consistent with these findings, IGV visualization of our acRIP-seq data revealed multiple ac4C peak alterations across the coding sequence (CDS) region of DYRK1A mRNA following NAT10 silencing (Updated Fig4.h).\nTo further assess whether NAT10 also regulates DYRK1A translation, we performed polysome profiling in senescent colonic epithelial cells. After ribosome fractionation, we examined the distribution of DYRK1A mRNA in polysome fraction. The results showed a marked reduction in polysome-associated DYRK1A mRNA in NAT10-knockdown cells (Response Fig.4a, Updated Fig.5f), suggesting a moderate decrease in translational efficiency.\nCollectively, these results support a model in which NAT10-mediated ac4C modification enhances both the stability and translation of DYRK1A mRNA. These newly obtained data have been incorporated into the revised manuscript to strengthen our mechanistic conclusions Revised results section (Line 241-246).\na\nPolysome\n*\nResponse Fig.4 qPCR analysis after polysome profiling ananlysis. a. Sucrose gradient fractionation of H-Sen NCM460 cells and H-Sen NCM460 cells with NAT10 knockdown. Relative DYRK1A mRNA abundance across polysome fractions was quantified by RT-qPCR. Data are presented as mean values ± S.D. Comparisons were performed by two-tailed t test. * p<0.05.\n6. While NAT10 is proposed as a therapeutic target, patient sample validation is lacking. Including clinical data would strengthen the study’s translational value.\nWe collected colonic mucosal biopsy specimens from healthy controls (HC), including both young and aged individuals, as well as from patients with ulcerative colitis (UC).\nIHC analyses were performed to assess the expression levels of NAT10 and DYRK1A (updated Fig.8a-c). Furthermore, in aged UC samples, we examined the correlation between NAT10 and DYRK1A expression (updated Fig.8d) and disease severity (updated Fig.8e), as determined by endoscopic Mayo scores. The results showed that higher protein expression levels of NAT10 and DYRK1A were associated with more severe disease activity in old-UC patients Revised results section (Line 344-346).\nInformation of human samples is provided in the Supplementary Table 4. Given the limited sample size in this study, we acknowledge that further validation in large, multi-\nFig8\ncenter cohorts will be essential to confirm and extend these findings.\na b\n10 **\n*\n8\n6\n4\n2\n0\nc d e\nYoun g ( - n H = C Y 1 o 0 u ) n g ( - n U = C 21 O ) l d ( - n H = C 12 O ) l d - ( U n C =16)\n** * **\n*\nUpdated Fig.8 NAT10 and DYRK1A expression are elevated in the aged colon\nand elderly UC tissues.\na-c, Representative IHC staining image and IHC score of NAT10 and DYRK1A in the colonic mucosa of young HC, Old-HC, young-UC and Old-UC patients. Scale bar, 50 μm.\nd, Correlation analysis between NAT10 and DYRK1A immunohistochemical scores in colonic mucosal biopsies from UC patients.\nerocs\ngniniats\n01TAN\nOld-HC\n01TAN\nA1KRYD\nYoung-HC Young-UC Old-UC\n10\n8\n6\n4\n2\n0\n0 2 4 6 8 10 NAT10 staining score\nerocs\ngniniats\nA1KYRD\nN = 37 10\nR = 0.71 P < 0.0001 8\n6\n4\n2\nerocs\ngniniats\nA1KYRD\n10\n8\n6\n4\n2\nerocs\ngniniats\n01TAN\ne, Comparison of NAT10 or DYRK1A immunohistochemical scores among old UC patients stratified by endoscopic Mayo score (Mayo0-1, Mayo2-3). n=6 for Mayo0-1 group, n=10 for Mayo2-3 group. Data are presented as mean values ± S.D. Spearman correlation analysis was used for (d). Student’s t-test was used for other analyses. ** p<0.01, * p<0.05.\n7. Do Remodelin and Harmine have similar effects in delay of colon senescence in aged mice? Which one is better?\nThank you for the insightful question. In this revised version, we additionally evaluated the effect of the DYRK1A inhibitor Harmine on colonic epithelial senescence in aged mice. We intraperitoneally administered Harmine (10 mg/kg, as previously reported6 ) to aged mice for 2 months (Updated Extended Data Fig.7a). In the colon, Harmine treatment led to a marked increase in Ki67-positive epithelial cells in the mucosa, indicating enhanced proliferative activity and mucosal regeneration (Updated Extended Data Fig.7b). We observed no signs of overt toxicity, supporting the safety of this regimen (Updated Extended Data Fig.7c-d).\nAlthough these two compounds target different nodes of the NAT10/DYRK1A axis, both of them could alleviate intestinal epithelial senescence, as supported by histological scoring, Ki67 staining, and evaluation of intestinal barrier function. Given the differences in physicochemical properties, dosage, and pharmacokinetic behavior between the two compounds, these factors may significantly influence their in vivo performance.\nBased on qPCR analyses of colonic tissues, the relative expression levels of p53, p21, and Lgr5 did not show significant differences in aged mice treated with Remodelin or Harmine for two months under the experimental conditions used in this study (Response Fig.5a-c). Our results highlight that both Nat10 and Dyrk1a are functionally targetable to modulate colonic aging.\nUpdated Extended Data Fig.7 Targeting NAT10/DYRK1A axis delays colon\nsenescence in mice.\na, The schematic diagram of the experimental procedure for Harmine or Vehicle treatment in mice. C reated in BioRender. Chen, J. (2026) https://BioRender.com/x16w\n213\nb, Immunofluorescence staining of Ki67 in aged mice treated with Harmine or vehicle. The mean fluorescence intensity of 10 fields was normalized to the vehicle group. Ki67+ cells were quantified from 15 random fields for comparison. Scale bar, 100 μm.\nc, Serum biochemical parameters (ALT, AST, BUN, CR, LDH, and CK-MB) in aged mice treated with Harmine or vehicle. ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CR, creatinine; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB isoenzyme.\nd, Hematoxylin-eosin staining of the heart, liver, spleen, l ung, and kidney from aged mice treated with Harmine or vehicle. Scale bar, 100 μm.\nelciheV\nenimraH\nHeart Liver Spleen Lung Kidney\nEH\ndegralnE\nFigS.7\nc\nns\nns ns\nns ns ns\nd\ne f\nNC+Vector shNat10+Vector shNat10+Dyrk1a\nIPAD/76iK\na b\nVehicle Harmine\nVehicle\n***\nHarmine(10mg/kg)\nNC+Vector shNat10+Vector shNat10+Dyrk1a\n#1 #2 #3 #1 #2 #3 #1 #2 #3 kDa\nNat10 135\nDyrk1a 100\n25\np21\np16 15\nβ-actin 40\na\n-9\n-10\n-11\n-12\n-13\nAged+R\nA\ne\ng\nm ed+Har mine\nResponse Fig.5 a-c, qPCR analyses of colonic tissues, the relative expression levels of p53, p21, and Lgr5 in aged mice treated with Remodelin or Harmine for two months.\n8. In Figure 4b, which sample's ac4C distribution is being shown here?Corresponding to Figure 4c, the distribution of all three samples should be included.\nIn the original Figure 4b, we presented the ac4C distribution from the mRNA in the control group, aiming to illustrate the baseline pattern of ac4C modification in this cell line. In the revised version, we have included the average ac4C distribution from three biological replicates for each group (Control, H-Sen, and H-Sen-Si) (Response Fig.6a), and these updated results are presented in the Updated Extended Data Fig.5a. For original Figure 4c, we have also added the enriched motifs of all three replicates from each group (Response Fig.6b, Updated Extended Data Fig.5b).\nResponse Fig.6 a, Position distribution of ac4C modification in protein coding genes in the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups from acRIP-seq data.\nnoisserpxe\nANRm\nevitaleR\n)nitca-β\not\ndezilamron\ntcΔ-(\n-6.0\n-6.5\n-7.0\n-7.5\n-8.0\nAged+R\nA\ne\ng\nm ed+Har mine\nnoisserpxe\nANRm\nevitaleR\n)nitca-β\not\ndezilamron\ntcΔ-(\n-1\n-2\n-3\n-4\n-5\nAged+R\nA\ne\ng\nm ed+Har mine\nnoisserpxe\nANRm\nevitaleR\n)nitca-β\not\ndezilamron\ntcΔ-(\np53 b p21 c Lgr5\nns\nns ns\na\nControl+NC-Si H-Sen+NC-Si H-Sen+NAT10-Si\nb\nControl+NC-Si H-Sen+NC-Si H-Sen+NAT10-Si\np=1e-10 p=1e-30 p=1e-20\np=1e-28 p=1e-27 p=1e-15\np=1e-30 p=1e-20 p=1e-20\nb, Enriched motifs of ac4C peaks in the control NC-Si, H-Sen NC-Si, and H-Sen NAT10-Si groups from acRIP-seq data\n9. In Figure 6b and 7k, Colonoids should be marked with a scale, and figure legends require specific scale bars.\nWe have now added scale bars to Figure 6b and Updated Fig.8a (Formerly Fig. 7k) to clearly indicate the magnification of the colonoid images as suggested. In addition, we have updated the corresponding figure legends to include specific scale bar information. These changes have been incorporated into the revised figures and legends.\n10. Lack of the letter l in Figure 7’s figure legends.\nWe have corrected the omission and added the missing label to the legend of Updated Fig.8d.\n11. Some methodological descriptions lack detail, such as the specific parameters for RNA ac4C modification assays. Some statistical methods are not clearly described, such as whether appropriate corrections were applied for multiple comparisons. Clarifying statistical procedures is highly recommended.\nWe have revised the Methods section and provided additional details regarding the RNA ac4C modification assays (Lines 520–530 and 550–560). Furthermore, we have clarified the statistical analysis procedures throughout the manuscript. Relevant revisions have been made in methods sections (Line673-678) and figure legends.\n12. Need grammar and language refinement: Some sentences would benefit from grammatical corrections and clearer phrasing. Examples include but not limited: Line 47: \"self-renew\" should be \"self-renewal\"; Line 56: \"inhibitors treatment with NAT10 or DYRK1A\" should be \"treatment with NAT10 or DYRK1A inhibitors\"; Line 58: \"rejuvenate intestinal aging\" would be clearer as \"rejuvenate the aged intestine\" or \"reverse intestinal aging\"; Line 148: \"a RNA ac4C-dependent manner\" should be \"an RNA ac4C-dependent manner”.\nWe have carefully revised the manuscript to address all the examples you listed:\n1.Line 47: \"self-renew\" changed to \"self-renewal\"\n2.Line 56: \"inhibitors treatment with NAT10 or DYRK1A\" changed to \"treatment with NAT10 or DYRK1A inhibitors\"\n3.Line 58: \"rejuvenate intestinal aging\" changed to \"rejuvenate the aged intestine\"\n4.Line 148: \"a RNA ac4C-dependent manner\" changed to \"an RNA ac4C-dependent manner\"\nIn addition, we have thoroughly proofread and edited the entire manuscript for grammar, clarity, and scientific style, with attention to tense consistency, article usage, and sentence structure. We believe the language is now significantly improved and appreciate your guidance in this regard.\nReviewer #2 (Remarks to the Author):\nThe authors of the submitted manuscript demonstrated that the NAT10/DYRK1A axis regulates the response during the late(old) onset of colitis in mice. In vitro and in vivo experiments showed that the levels of NAT10 regulate colonic senescence status, and the NAT10 stabilizes DYRK1A mRNA. In summary, it is an interesting concept, and the authors provided plenty of data to support their hypothesis. I have several comments that I would like the authors to address.\n1.Figure 3e. IF, Occludin, please include the colonic crypts that show the entire crypt. Due to the angle of the tissues, we do not see the full crypt (specifically the crypt included in the center of the image).\nWe sincerely thank the reviewer for pointing out this issue. We have carefully re-examined and updated the image in Figure 3e (Occludin staining) to better show complete colonic crypt structures, including the central region (Response Fig.7a, updated Fig.3.e). If there are any remaining concerns regarding image quality or interpretation, we would greatly appreciate further suggestions.\na\nResponse Fig.7 a, Immunofluorescence staining of Ocln in colon tissues from Young-WT, Aged-WT, and Aged-Nat10+/- mice groups.\n2.Figure 3e. IF LGR5, I am unsure whether the author noticed that the stem cell marker staining is in multiple crypt sections while it should be localized to the bottom. TW-gnuoY\nTW-degA\n-/+01taN-degA\nOcln/DAPI\nSubmitted Revised\nAs such, this image is misleading; not everybody works for Western, IF, etc. The authors should include in situ hybridization instead.\nResponse:\nThank you for this important point. Based on our Western blot analysis, Lgr5 protein expression was indeed reduced in aged colonic tissue. Detecting Lgr5 by immunofluorescence in the colon is more challenging than that in the small intestine, likely due to lower endogenous expression levels, technical limitations in antibody sensitivity. To address this concern, we attempted fluorescence in situ hybridization (FISH) to visualize Lgr5 expression in colonic tissues (Response Fig.8a). Although the results showed a similar trend, reduced Lgr5 expression localized to the crypt base in aged mice. However, the signal was relatively weak and not sufficiently robust for quantitative analysis\nLgr5/DAPI\nIF FISH 8\n6\n4\n2\nA\n-\nW\nge\nT\nd -\nNAT10+/-\n.\nResponse Fig.8 a, Immunofluorescence staining and Fish detection of Lgr5 in colon tissues from Young-WT, Aged-WT, and Aged-Nat10+/- mice groups.\nGiven the limitations in imaging-based detection, we have chosen to retain only the Western blot results in Fig. 3. We sincerely thank the reviewer for highlighting this point, which helped us further strengthen the rigor and interpretation of our findings.\n3.The authors delivered Remodelin via IP; however, it is an orally available drug. Please explain.\nnoisserpxe\nANRm\nevitaleR\nLgr5\nTW-gnuoY\nTW-degA\n-/+01taN-degA\na b\nWhile some studies have administered Remodelin orally or via gavage, intraperitoneal (IP) injection was also employed, which was effective and well-tolerated in mouse models1,5,7. In our study, to achieve the required dosage within the limited injection volume, we prepared a concentrated stock solution using solubilizing agents (DMSO, PEG300, and Tween-80) (Revised manuscript Line 634-643). We were concerned that oral administration might irritate the gastrointestinal mucosa, particularly in aged or colitis-prone mice, thereby confounding key readouts such as colonic inflammation and epithelial barrier integrity. Therefore, IP injection was selected as the delivery route in our study.\n4.Figure 7 and extended Figure 7: please provide weight information throughout the experiments with clinical and histological scores.\nIn the revised manuscript, we have included body weight measurements throughout the colitis experiments, along with quantitative clinical and histological scores. These data are presented in upadted Fig 7b,e,m,n and upadted Extended Data Fig.9b,d, with corresponding descriptions added to the Results section and figure legends.\nVehicle Rem\n#1 #2 #3 #1 #2 #3\nDyrk1a 100\nγ-H2AX 15\nβ-actin 40\nUpdated Extended Data Fig.9 Pharmacological inhibition of NAT10 or DYRK1A alleviates DSS-induced colitis in aged mice.\nelciheV\nmeR\nPAS γ-H2AX/DAPI\n** **\nac4C\nMB\nFigS.8\na b c\nVehicle\nRem\n**\nVehicle ***\nRem\nd e\nf g Vehicle Harmine\n**\nD7 D11 D7 D11 ns\nb, Monitoring of body weight changes throughout the process of DSS-induced acute colitis in 18-mon mice treated with Rem or vehicle.\nd, Representative HE staining and γ-H2AX immunofluorescence staining images in 18-mon mice following DSS exposure and treatment with Rem or vehicle. n=5. The number of γ-H2AX+ cells from 15 fields were analyzed.\na b c\nUpdated Fig.7 Deficiency of Nat10 in aged mice alleviates DSS-induced colitis\nb, Monitoring of body weight changes throughout the process of DSS-induced acute colitis in aged (12-mon) Nat10-WT and Nat10+/- mice. n=5.\nTW\n-/+01taN\n***\nW\nN\nT at10+/-\ng γ-H2AX/DAPI Ki67/DAPI PAS h\n*** ***\n***\nac4C\nMB\nd e f\nWT Nat10+/-\nWT\n***\n*** Nat10+/-\nWT\nNat10+/-\ni j k\nWT Nat10+/- Dyrk1a\nkDa\nγ-H2AX 15\nOcln 75\n* Dyrk1a 100 **\n** ** *** Nat10 130\nβ-actin DSS 40\n11D\nFig7\n**\nColonscopy\n***\nl n\nNC+Vector 15\nm\n10\n5\nerocs\nlacigolotsiH\n***\n**\n** ns\n120\n110\n100\n90 80 70 01234567891011\n)%(egnahc\nthgiew ydoB\nshNat10+Vector shNat10+Dyrk1a\nAAV-NC+AAV-Vector\nAAV-ShNat10+AAV-Vector AAV-ShNat10+AAV-Dyrk1a\nDays\n3%DSS Tap water\ne, Pathological score of colonic mucosa from aged Nat10-WT and Nat10+/- mice following DSS-induced colitis. n=5.\nm, Monitoring of body weight changes throughout the process of DSS-induced colitis after 2-month AAV tail-vein delivery (NC+Vector, shNat10+Vector, or shNat10+Dyrk1a) in aged mice. n=5.\nn, Representative hematoxylin–eosin staining and pathological scores of colonic mucosa from aged mice treated with DSS following 2-month AAV tail-vein delivery (NC+Vector, shNat10+Vector, or shNat10+Dyrk1a). Scale bar, 100 μm. n=5. Data are presented as mean values ± S.D. Comparisons were performed by Student’s t-test. Body weight changes over time were analyzed by two-way ANOVA with Tukey’s multiple comparison. *** p<0.001, ** p<0.01, * p<0.05.\n5.Notably, the authors show that aged-Nat10+/- mice have increased levels of Ki67 (Figure 3e), which suggests that they will be more susceptible to the injury (as proliferative cells are more susceptible to the injury); however, the results are the opposite. Please explain\nThank you for this thoughtful comment. Indeed, proliferative epithelial cells are generally considered more vulnerable to acute injury due to their active cell cycle state. However, in the colon, proliferative epithelial cells are largely confined to the base of the crypts, where they reside in a relatively protected niche supported by stromal, immune, and microbiota-derived signals. These proliferative cells continuously give rise to differentiated epithelial cells that migrate upward to maintain the mucosal barrier. During aging or upon inflammatory challenge, the capacity for crypt-base proliferation becomes critical for epithelial repair and mucosal restoration. Several studies have demonstrated that enhanced epithelial proliferation (reflected by increased Ki67⁺cells) promotes epithelial regeneration, attenuates mucosal injury, and improves recovery following inflammation or epithelial damage8-10.\nIn our study, we observed that NAT10 knockdown alleviated epithelial senescence and enhanced proliferative capacity across multiple models (including in vitro colonic epithelial cells, in vivo aged mice, and colonoid cultures). Notably，Ki67 expression was moderately increased in aged Nat10+/− mice, reflecting an enhanced regenerative response or preservation of proliferative capacity. This basal proliferative activity may contribute to improved mucosal repair and epithelial turnover, thereby protecting the colon from “leaky” gut. We have clarified these interpretations in the discussion section to emphasize the physiological relevance of enhanced epithelial regeneration in the context of NAT10 deficiency (Revised manuscript Line 388-392).\n6.Figure 7k: Please include IHC stains from tissues of healthy young and aged patients (non-IBD).\nWe have now included IHC staining of NAT10 and DYRK1A in colonic biopsy samples from healthy young and aged non-IBD individuals (updated Fig. 8a-c). Both NAT10 and DYRK1A exhibited elevated expression in the colonic epithelium of aged human individuals. These results further support the age-associated upregulation of the NAT10/DYRK1A axis in the colon and underscore the potential translational relevance of our findings. Information of human samples is provided in the Supplementary Table 4.Given the limited sample size, we acknowledge that further validation in large, multi-center cohorts will be essential to confirm and extend these findings.\nFig8\na b\n10 **\n*\n8\n6\n4\n2\n0\nc d e\nYoun g ( - n H = C Y 1 o 0 u ) n g ( - n U = C 21 O ) l d ( - n H = C 12 O ) l d - ( U n C =16)\n** * **\n*\nUpdated Fig.8 NAT10 and DYRK1A expression are elevated in the aged colon\nand elderly UC tissues.\nerocs\ngniniats\n01TAN\nOld-HC\n01TAN\nA1KRYD\nYoung-HC Young-UC Old-UC\n10\n8\n6\n4\n2\n0\n0 2 4 6 8 10 NAT10 staining score\nerocs\ngniniats\nA1KYRD\nN = 37 10\nR = 0.71 P < 0.0001 8\n6\n4\n2\nerocs\ngniniats\nA1KYRD\n10\n8\n6\n4\n2\nerocs\ngniniats\n01TAN\na-c, Representative IHC staining image and IHC score of NAT10 and DYRK1A in the colonic mucosa of young HC, Old-HC, young-UC and Old-UC patients. Scale bar, 50 μm.\nd, Correlation analysis between NAT10 and DYRK1A immunohistochemical scores in colonic mucosal biopsies from UC patients.\ne, Comparison of NAT10 or DYRK1A immunohistochemical scores among old UC patients stratified by endoscopic Mayo score (Mayo0-1, Mayo2-3). n=6 for Mayo0-1 group, n=10 for Mayo2-3 group. Data are presented as mean values ± S.D. Spearman correlation analysis was used for (d). Student’s t-test was used for other analyses. ** p<0.01, * p<0.05.\n7.Please follow the nomenclature of murine and human genes and proteins and proofread your manuscript. There are multiple minor grammatical and orthographical mistakes.\nWe have thoroughly proofread and revised to adhere to the standard nomenclature for murine and human genes and proteins (e.g., mouse gene Nat10, protein Nat10; human gene NAT10, protein NAT10), and to correct all grammatical and typographical errors. We have also carefully checked gene and protein names throughout the main text, figures, and legends to ensure consistency and accuracy.\nReviewer #4 (Remarks to the Author):\nUsing intestinal models, both in vitro and in vivo, this study investigates the role of NAT10 in senescence. Their data suggest that NAT10 is upregulated during senescence, contributing to the phenotype through ac4C modification of DYRK1A mRNA in culture. Interestingly, the NAT10/DYRK1A axis is also upregulated in UC models in humans and mice. In Nat10+/- mice, Nat10 levels are substantially reduced: Using this model, they show the critical role of this axis in the colitis of aged mice. The age-associated alteration in the NAT10/DYRK1A axis is potentially interesting, but the link to senescence is weak.\n1、Fig. 1: Overall, the senescence assay is not robust. Are these cell models well-established (no references are provided)? With a quick search, I was not able to find previous studies characterizing senescence in these cell lines. Sa-b-gal is a good marker, but it is not necessary or sufficient for defining senescence. Validation of persistent cell cycle arrest is essential. In addition, it would be helpful if they added blots of p16, a more specific senescence marker (Fig. 1e).\nThank you for this thoughtful comment. We apologize for the lack of clarity in the original manuscript regarding the establishment and validation of our senescence models. In the revised version, we have now expanded the methods section to better describe the rationale and optimization process for inducing senescence in human colonic epithelial cells. Both NCM460 and CCD841 have been previously reported in the literature to display senescent phenotypes: NCM460 has been used to study oncogene-induced senescence (OIS)11 ; CCD841 has been applied in etoposide-induced senescence models to explore the molecular mechanisms underlying intestinal epithelial senescence12.\nTo further validate senescence in our models, we examined the expression of p16 and Cyclin D1 by Western blotting. In both H-Sen and D-Sen cells, p16 expression was elevated, whereas Cyclin D1 expression was markedly reduced (Response Fig.9a, updated Fig.1e). Moreover, knockdown of NAT10 using two independent siRNA sequences significantly decreased p16 expression while restoring Cyclin D1 levels (Extended Data Fig.2a). In addition, overexpression of NAT10-WT, while not NAT10-MUT, could partially reverse the downregulation of p16 and upregulation of Cyclin D1 and the positive percentage of EdU (Response Fig.9b-d, updated Fig.2b and updated Extended Data Fig.2d,e).\nc d\n50\n40\n30\n20\n10\nResponse Fig.9 NAT10 regulates colon epithelial cell senescence phenotypes through mRNA ac4C modification\na, Western blot of Cyclin D1 and p16 in senescent NCM460 and CCD841 cells.\nb, Western blot of CyclinD1 and p16 in NAT10-reexpressed (NAT10-WT or NAT10-MUT) cells after transfected with NAT10-Si and treated with H2O2 or DOX.\nc-d, Flow cytometric analysis of EdU-positive cells in NAT10-reexpressed (NAT10-WT or NAT10-MUT) NCM460 cells following NAT10 siRNA transfection and senescence induction by H₂O₂ or DOX. Data are represented as mean ± SD with individual data points shown (n=3). Comparisons were performed by two-tailed t test. ** p<0.01, ns p>0.05.\n2、In the doxycycline model, they treated the cells for 72 h. I assume they conducted senescence assays at this point. If so, it may be too early. Typically, cells are maintained for additional days without drugs to fully establish senescence. Of course, this can vary depending on the cell type, which is why a comprehensive characterization of senescence is essential before correlating it with other phenotypes, such as an mRNA modification. It would also be helpful if the authors could briefly describe the H2O2 model.\nThank for this important point. Since doxorubicin (DOX) is known to exert transcriptional inhibitory effects, senescence assessments were not performed immediately after the 72-hour DOX treatment. Instead, the cells were maintained for an additional 5 days after DOX withdrawal before senescence assays were conducted. This approach is consistent with our previously published protocol for D-Sen MEF cells (Chen et.al. Nat Aging, 2021)13.\nAs for the H O ‐induced senescence model (H-Sen), we adapted and modified, protocols previously reported in colorectal cancer cell lines (e.g. SW480, CT26)14 as well as human fibroblasts15, and optimized the conditions specifically for normal colonic epithelial cells. In our system, NCM460 cells were first exposed to 250 μM H O for 48 hours, followed by continued incubation in medium containing 50 μM H O for an additional 6 days to allow the establishment of a stable senescent state. The H₂O₂-containing medium was replaced daily throughout the induction period. We have now revised the Methods section to clearly specify this timeline and prevent further confusion (Line411-422).\n3、These senescence assays are all under the assumption that these cells are resistant to cell death. Or are we looking at residual cells surviving the drug treatment?\nResistance to apoptosis is a hallmark of senescent cells. In our initial experiments, we confirmed that 300 nM DOX and 250 μM H O are sublethal doses for NCM460 cells, as these treatments did not significantly induce apoptosis (Response Fig.10a). In addition, cell senescence was successfully induced, as evidenced by an increased proportion of SA-Gal positive cells (updated Fig1.b), elevated expression of multiple senescence markers (p53, p21, p16, and γ-H2AX), reduced Cyclin D1 protein levels (updated Fig1.e), and a higher proportion of cells arrested in the G1 phase (Response Fig. 10b,c, ).\na\nns ns\nb c\nNCM460 CCD841\n******\n*** ***\nResponse Fig.10\na, Apoptosis flow cytometry analysis in Control, H-Sen and D-Sen NCM460 cells.\nb-c, Cell cycle distribution and the proportion of G1-phase cells in proliferating and senescent NCM460 and CCD841 cells were assessed by flow cytometry following PI staining. Data are represented as mean ± SD with individual data points shown (n=3). Comparisons were performed by two-tailed t test. *** p<0.001, ns p>0.05.\nAfter senescence induction, we transfected NAT10-targeting siRNAs into these senescent cells for 48h and found that NAT10 knockdown reduced cell viability as measured by a redox-based assay, and increased caspase-3/7 activity and apoptosis in senescent cells (updated Fig2. g,h), highlighting NAT10 as a potential senolytic target in senescent colonic epithelium.\n4、Fig. 2 suggests that the knockdown of NAT10 promotes apoptosis rather than bypassing senescence during dox treatment, even though overall p53 signaling and DDR are alleviated. I wonder what could be a mechanism for this? Is the cell cycle maintained (western for cell cycle factors, BrdU/EdU assays would be helpful)?\nAs suggested, we further examined the proportion of EdU-positive cells and the expression of Cyclin D1 after senescence induction. We found that NAT10 knockdown increased Cyclin D1 expression and the percentage of EdU-positive cells (updated Fig2.b and updated Extended Data Fig.2d,e). Similarly, in the late-passage CCD841 cell line (P15), silencing NAT10 also increased the EdU-positive cell populations. We appreciate the reviewer’s insightful suggestion, and these results further support the regulatory role of NAT10 in cellular senescence.\nDYRK1A, one of the downstream target genes of NAT10, has previously been reported to regulate cell cycle progression and promote the accumulation of DNA damage. In line with these findings, we speculate that elevated DYRK1A expression in senescent epithelial cells may contribute to sustained DDR. To further validate the functional link between NAT10 and DYRK1A, we performed rescue experiments in vitro using both NCM460 and CCD841 cells. The results confirmed that the senescence-associated phenotypes mediated by NAT10 are dependent on DYRK1A. We have elaborated on these findings and their mechanistic implications in the revised Discussion section of the manuscript (Revised manuscript Line 366-371, 383-385).\n5、Senescence is generally associated with 'persistent' DDR, which is rather modest. However, Fig. 2c suggests a highly robust DDR in these cells. this is perhaps consistent with my earlier comment on the timing of Dox-senescence (72h might be too early). Is it possible that the phenotype is associated with DDR rather than senescence?\nWe appreciate the reviewer's insightful question regarding the relationship between the observed DDR and senescence. We agree that senescence is typically associated with a persistent DDR. In our senescence model in colonic epithelial cells, we indeed observed a significant increase in γ-H2AX levels, as detected by both Western blot and immunofluorescence.\nBased on the literature, we consider that the extent of DNA damage response (DDR) during senescence may vary across different cell types. For example, human fibroblasts tend to exhibit relatively mild DDR upon senescence. In contrast, a recent study reported that DOX-induced senescence in Caco-2 colon epithelial cells is accompanied by a pronounced increase in DDR levels (Response Fig.11).\nResponse Fig.11. 𝛾-H2AX immunofluorescence staining in Caco-2 colon epithelial cell (Z. Cao.et.al，Adv. Sci. 2025, DOI：10.1002/advs.202515794)\nAlthough DNA damage is recognized as an early and important event in the induction of cellular senescence, it may not be sufficient on its own to trigger the full senescence program. Our data, based on multiple senescence indicators (including p53, p21, p16, and SA-Gal staining), provide comprehensive evidence that the regulatory role of NAT10 in senescence extends beyond its effects on the DDR.\n6、Fig. 3: How about cell death? Do the authors observe enhanced cell death in aged Nat10+/- intestine? This might be more pronounced in the colonoid experiments, particularly in the presence of LPS (Fig. 6)?\nWe found that NAT10 knockdown did not significantly affect apoptosis in proliferating colonic epithelial cells. However, NAT10 knockdown could increase apoptosis in already senescent cells (Response Fig.12 a,b).\nTo further evaluate cell death in vivo, we performed TUNEL staining and cleaved caspase-3 immunohistochemistry on colonic tissues from aged Nat10⁺/⁻ and aged WT mice (Response Fig.12c). Indeed, in the cross-sectional analysis of colonic tissues from aged Nat10⁺/⁻ mice, we observed relatively fewer dying cells compared with aged WT mice. We previously found that aged Nat10⁺/⁻ mice exhibit enhanced crypt regenerative capacity, as indicated by increased Ki67 and Lgr5 expression. Taken together, these findings suggest that both sustained proliferative capacity and removal of damaged cells may contribute to the attenuated colonic aging phenotype observed in aged Nat10⁺/⁻ mice.\na Control+NC-Si Control+NAT10-Si1 Control+NAT10-Si2 20\n15\n10\n5\nb\nH-Sen+NC H-Sen+NAT10-Si1 H-Sen+NAT10-Si2\nc\nIHC:C-Casepase3 Tunel/DAPI\nAged-WT Age-Nat10+/- Aged-WT Age-Nat10+/-\nResponse Fig.12\na, Apoptosis flow cytometry analysis in Control NCM460 cells after transfected with NAT10-SiRNAs.\nb, Apoptosis flow cytometry analysis in H-Sen NCM460 cells after transfected with NAT10-SiRNAs.\nc, immunohistochemistry of cleaved caspase-3 and Immunofluorescence staining of Tunel in the colons of Aged-WT, and Aged-Nat10+/- mice. Data are represented as mean\n)%(sllec\nevitisop\nV\nnixennA\nns\nns\n25\n20\n15\n10\n5\n)%(sllec\nevitisop\nV\nnixennA\n**\n**\n± SD with individual data points shown (n=3). Comparisons were performed by two-tailed t test. ** p<0.01, ns p>0.05.\n7、Fig. 4 (acRIP-seq): I couldn't find information on the number of biological replicates. How consistent are the replicates? Also, it would be helpful if they provided more general information, such as the total number of peaks identified in each condition, particularly considering that only a small number of differential peaks were identified (Fig. 4e).\nThanks for the valuable suggestions. We performed acRIP-seq on three biological replicates per group (Control, H O -induced senescent cells, and NAT10 knockdown in senescent cells), and have now provided detailed descriptions of the dataset and analyses in the revised manuscript. A summary of peak numbers and ac4C-enriched gene counts for each sample is presented in the revised Response Table 1.\nFrom the acRIP-seq analysis, 1,228 hyper-acetylated peaks (log₂FC>0) corresponding to 858 genes were identified in the H-Sen group compared with the control group (supplementary dataset1). In contrast, 640 hypoacetylated peaks (log₂FC<0) corresponding to 529 genes were detected in the H-Sen NAT10-Si group compared with the H-Sen group (supplementary dataset2). The corresponding information updated in the Revised results section (Line 193-196).\nTo pinpoint key transcripts regulated by NAT10, we applied filtering criteria. The complete list of differentially modified peaks and their corresponding genes has been provided in the supplementary materials. We first identified 32 protein-coding transcripts that exhibited increased ac4C abundance (log₂FC>1.5) in senescent cells (H-Sen) compared to control. Upon NAT10 knockdown in senescent cells (H-Sen-Si), 11 protein-coding transcripts showed reduced ac4C abundance (log₂FC>1.5). The intersection of these datasets yielded four candidate target genes: THAP5, DYRK1A, LMLN, and MVB12A (updated Fig.4d). We merged the three biological replicates for each acRIP-seq group and performed IGV visualization, as shown in updated Extended Data Fig.5d.\nWe further analyzed the FPKM values of input samples across the three biological replicates in the acRIP-seq dataset. The FPKM distributions showed good consistency and correlation among replicates Response Fig.13a,b.\nResponse Fig.13 FPKM distributions and correlation analysis of input samples across the three biological replicates in the acRIP-seq.\n8、Fig. 5e: No details on the reporter construct are provided. Also, it lacks positive controls.\nIn our luciferase reporter assay, we cloned the DYRK1A promoter region (−2000 to +100 bp relative to the transcription start site) into the pGL4.10 firefly luciferase vector. The Renilla luciferase plasmid pRL-TK was co-transfected for normalization. We repeated the experiment and have now included a positive-control group using a firefly luciferase plasmid driven by the CMV promoter, which drives robust firefly luciferase activity and confirms that the reporter system is functioning properly. This information has been added to the Methods section (Revised manuscript Line 438-447), and the new result has been included in the revised manuscript (updated Extended Data Fig.5e)\n9、Fig. 7f: The authors tested SASP genes here. Do they mean that these cytokines are expressed by 'senescent' epithelial cells? Or are they expressed by immune cells? Are these SASP genes also upregulated during ageing with no DSS (e.g., Fig. 3, 6)?\nIL-1β, IL-6, and TNF-α are widely recognized as key indicators of intestinal inflammation in murine colitis models16. Moreover, accumulating evidence in the field of intestinal aging indicates that IL-1β, IL-6, and TNF-α are also important systemic inflammatory mediators whose expression increases in the colonic tissues of aged mice12,17-19. However, IL-1β, IL-6, and TNF-α can indeed originate from multiple cellular sources. It has been reported that senescent colonic epithelial cells, fibroblasts, and immune cells such as macrophages are all capable of secreting these cytokines16,20. In our Figure 7, we observed that following DSS-induced colitis, the expression of senescence markers such as γ-H2AX and P21 in colonic tissues was reduced. Consistently, we also detected decreased levels of IL-1β, IL-6, and TNF-α. However, we acknowledge that referring to these cytokines as SASP factors in this context is not precise. We have revised the relevant statements in the revised manuscript accordingly (Revised manuscript Line322-323).\nWe measured the expression of IL-1β, IL-6, and TNF-α in the colonic tissues of 2-month-old and 18-month-old mice, and found that the mRNA levels of these inflammatory cytokines were markedly increased in the colons of 18-month-old mice (Updated Extended Data Fig.3b). Moreover, compared with aged WT mice, the expression of IL-1β, IL-6, and TNF-α was reduced in the colonic tissues of Aged-Nat10⁺/⁻ mice (Updated Extended Data Fig.3d).\na b\n**\nc\nUpdated Extended Data Fig.3. Nat10 and RNA ac4C modifications are upregulated in the colon of aged mice.\nb, qPCR analysis of Il-1β, Il-6 and TNF-α mRNA expression in colon of 2-mon and 18-mon mice. n=5.\nd, qPCR analysis of Il-1β, Il-6 and TNF-α mRNA expression in Aged-WT and Aged-Nat10+/- mouse colon. n=5 for Aged-WT group; n=7 for Aged-Nat10+/- group. Data are presented as mean values ± S.D. Comparisons were performed by two-tailed t test. *** p<0.001, ** p<0.01, * p<0.05.\n10、RNAi experiments: it is highly encouraged to test multiple target sequences to alleviate off-target effects.\nWe used two independent siRNAs to knock down NAT10 expression and found that both effectively attenuated DOX- or H2O2-induced cell senescence, as evidenced by reduced percentages of SA-Gal-positive cells (Response Fig. 14a,b), decreased expression of senescence-associated marker p16, and increased Cyclin D1 levels (Updated Extended Data Fig.2a,b). NAT10-Si2 was used for subsequent rescue experiments involving re-expression of NAT10-WT or NAT10-MUT.\nSimilarly, in the CCD841 cell line, knockdown of either NAT10 or DYRK1A in P15 cells using two distinct siRNAs also reduced the proportion of SA-β-Gal-positive cells, BM\nC4ca\nColonic inflammatory markers\n2-mon 5\n4\n18-mon 3 ** ** **\n2-mon 2\n1\n18-mon 0\nIL-1 IL-6 TNF-\nColonic inflammatory markers\nγ-H2AX/DAPI\nAged-WT Aged-Nat10+/-\n** ** *\n***\nnoisserpxe\nANRm\nevitaleR\n2-mon\n18-mon\nd\nincreased the proportion of EdU-positive cells (Response Fig. 14c-h, Updated Extended Data Fig.6d-f), and enhanced colony-forming ability (Response Fig. 14i).\nResponse Fig.14\na, Western blot of NAT10 in NCM460 cells after transfected with NAT10-SiRNAs and treated with H O or DOX.\n2 2\nb, Analysis of SA-Gal activity in NCM460 cells after transfected with NAT10-SiRNAs and treated with H O or DOX.\n2 2\nc, Western blot of NAT0 in P15 CCD841cells after transfected with NAT10-SiRNAs.\nneS-H\nneS-D\nNC-Si NAT10-Si1 NAT10-Si2 120\n90\n60\n30\n)%(llec\n+laG-AS\nNC-Si\nNAT10-Si1\nNAT10-Si2\nNC-Si\nNAT10-Si1\nNAT10-Si2\nCCD841-P15\niS-CN\n1iS-01TAN\n2iS-01TAN\nNC-Si NAT10-S\nN\nI1 AT10-Si2 NC-Si NAT10-S\nN\nI1 AT10-Si2\nNAT10\nβ-actin\nH-Sen NCM460 D-Sen NCM460\nNAT10-S\nN\nI1 AT10-Si2\n60\nβ-actin 20\nCCD841-P15\n)%(llec\n+laG-AS\n30\n20\n10\negatnecrep\nllec\nevitisop\nUdE\na b\n*** ***\n*** ***\nc d e\n**\n**\n** **\nNC-Si DYRK1A D\n-S\nY\ni1 RK1A-Si2\nCCD841-P15 CCD841-P15\nNC-Si\nNAT10-Si1 NAT10-Si2\nNC-Si\nDYRK1A-Si1 DYRK1A-Si2\nCCD841-P15 CCD841-P15\niS-CN\n1iS-A1KRYD2iS-A1KRYD\n30\nDYRK1A DYRK1A-Si1 10\nβ-actin\nDYRK1A-Si2 0\nD N Y C R - K S D 1 i A Y - R S K i1 1A-Si2\negatnecrep\nllec evitisop\nUdE\nf g h\n**\n**\n** **\ni\nd, Analysis of SA-β-Gal activity in P15 CCD841cells after transfected with NAT10-SiRNAs.\ne, Flow cytometric analysis of EdU-positive cells in P15 CCD841cells after transfected with DYRK1A-SiRNAs.\nf, Western blot of NAT0 in P15 CCD841cells after transfected with NAT10-SiRNAs.\ng, Analysis of SA-β-Gal activity in P15 CCD841cells after transfected with DYRK1A-SiRNAs\nh, Flow cytometric analysis of EdU-positive cells in P15 CCD841cells after transfected with DYRK1A-SiRNAs\ni, Colony-forming analysis in P15 CCD841cells after transfected with NAT10 or DYRK1A-SiRNAs.\n11、Nat10 knockout mice: It would be helpful if the authors could provide more details, such as the targeting strategy, in the Methods section.\nThanks for pointing out this missing information in our manuscript. we have now included additional details regarding the Nat10 knockout strategy in the revised Methods section. Briefly, the Nat10+/− mice used in this study were generated by deleting exon 4 using a CRISPR/Cas9-based approach (Response Fig.15). Genotyping was performed by PCR using specific primers flanking the deleted region. We have updated the relevant section in the Methods to clearly reflect these experimental details.\nResponse Fig.15 CRISPR/Cas9-based approach to produce Nat10 knockout mice.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nWe have carefully revised the manuscript in accordance with the comments provided.\nif you have any further suggestions, we would be happy to further refine our work based on your suggestions.\nReference\n1. Zhang, S. et al. NAT10-mediated mRNA N(4)-acetylcytidine reprograms serine metabolism to drive leukaemogenesis and stemness in acute myeloid leukaemia. Nat Cell Biol 26, 2168-2182 (2024).\n2. Yu, C. et al. NAT10 promotes vascular remodelling via mRNA ac4C acetylation. Eur Heart J 46, 288-304 (2025).\n3. Liu, D. et al. NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis. Ann Rheum Dis 83, 1118-1131 (2024).\n4. Balakrishnan, M.P. et al. THAP5 is a human cardiac-specific inhibitor of cell cycle that is cleaved by the proapoptotic Omi/HtrA2 protease during cell death. Am J Physiol Heart Circ Physiol 297, H643-653 (2009).\n5. Shi, J. et al. NAT10 Is Involved in Cardiac Remodeling Through ac4C-Mediated Transcriptomic Regulation. Circ Res 133, 989-1002 (2023).\n6. Wang, P. et al. A high-throughput chemical screen reveals that harmine-mediated inhibition of DYRK1A increases human pancreatic beta cell replication. Nat Med 21, 383-388 (2015).\n7. Xie, R. et al. NAT10 Drives Cisplatin Chemoresistance by Enhancing ac4C-Associated DNA Repair in Bladder Cancer. Cancer Res 83, 1666-1683 (2023).\n8. Ren, X. et al. DHX9 maintains epithelial homeostasis by restraining R-loop-mediated genomic instability in intestinal stem cells. Nat Commun 15, 3080 (2024).\n9. Bao, W. et al. Inhibiting sorting nexin 10 promotes mucosal healing through SREBP2-mediated stemness restoration of intestinal stem cells. Sci Adv 9, eadh5016 (2023).\n10. Gonzalez, E.A. et al. TET3-mediated DNA oxidation is essential for intestinal epithelial cell response to stressors. Proc Natl Acad Sci U S A 120, e2221405120 (2023).\n11. Henriques, A.F., Barros, P., Moyer, M.P., Matos, P. & Jordan, P. Expression of tumor-related Rac1b antagonizes B-Raf-induced senescence in colorectal cells. Cancer Lett 369, 368-375 (2015).\n12. Hu, X. et al. Intestinal Epithelial Cell-specific Deletion of Cytokine-inducible SH2-containing Protein Alleviates Experimental Colitis in Ageing Mice. J Crohns Colitis 17, 1278-1290 (2023).\n13. Chen, S. et al. Bifidobacterium adolescentis regulates catalase activity and host metabolism and improves healthspan and lifespan in multiple species. Nat Aging 1, 991-1001 (2021).\n14. Choi, Y.W. et al. Senescent Tumor Cells Build a Cytokine Shield in Colorectal Cancer. Adv Sci (Weinh) 8, 2002497 (2021).\n15. Tollefsbol, T.O. Biological Aging: Methods and Protocols. Methods in Molecular Biology 1048 (2013).\n16. Neurath, M.F. Cytokines in inflammatory bowel disease. Nat Rev Immunol 14, 329-342 (2014).\n17. Hohman, L.S. & Osborne, L.C. A gut-centric view of aging: Do intestinal epithelial cells contribute to age-associated microbiota changes, inflammaging, and immunosenescence? Aging Cell 21, e13700 (2022).\n18. Meng, G., Monaghan, T.M., Duggal, N.A., Tighe, P. & Peerani, F. Microbial-Immune Crosstalk in Elderly-Onset Inflammatory Bowel Disease: Unchartered Territory. J Crohns Colitis 17, 1309-1325 (2023).\n19. Wellman, A.S. et al. Intestinal Epithelial Sirtuin 1 Regulates Intestinal Inflammation During Aging in Mice by Altering the Intestinal Microbiota. Gastroenterology 153, 772-786 (2017).\n20. Neurath, M.F. Strategies for targeting cytokines in inflammatory bowel disease. Nat Rev Immunol 24, 559-576 (2024)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed the majority of my previous concerns, and significantly improved the manuscript.\nThere are still some questions need to be addressed:\n1. For the acRIP-seq data, why the authors used log₂FC>1.5 as a threshold?\n2. Fig. 3g needs quantification.\n3. The original data of Western blotting should be included.", "reviewer_2": "The authors satisfactorily addressed my comments.", "reviewer_4": "The authors have addressed most of my questions.\nA minor point remains regarding the reporter system (#8). They have added details about the construct, which is helpful.\nHowever, I would like to clarify my original question about “positive controls.” They included a CMV-driven luciferase (a technical control), but what I had in mind was a biological positive control, i.e., a condition known to induce DYRK1A transcription (if such a condition available). This is particularly important given that the conclusion is based on a negative result. Therefore, the insights from this data (ED Fig. 5e) in its current form are limited. Nevertheless, given the other supportive data (e.g., mRNA stability), I feel this could be a minor point."}, "author_response": "Re: Targeting NAT10 Alleviates Colonic Senescence and Elderly-Onset Colitis by Disrupting RNA Acetylation-Mediated DYRK1A Stabilization\nNature Communications Reference: NCOMMS-25-11798\nReviewer #1 (Remarks to the Author):\nThe authors have addressed the majority of my previous concerns, and significantly improved the manuscript.\nThere are still some questions need to be addressed:\nWe sincerely appreciate your constructive comments, which have significantly improved the clarity and rigor of our work.\n1. For the acRIP-seq data, why the authors used log₂FC>1.5 as a threshold?\nResponse:\nThank you for pointing out this issue. In our acRIP-seq dataset, a large number of transcripts exhibited modest changes in ac4C signal (log₂FC > 0; Lines 193–196). However, given the semi-quantitative nature of enrichment-based assays and the variability introduced by immunoprecipitation efficiency, we applied a fold-change cutoff to reduce background noise and focus on high-confidence candidates. The use of a stringent fold-change threshold is a commonly adopted strategy in RNA modification–RIP sequencing, including acRIP-seq1-3, for prioritizing downstream candidate genes.\nThe log₂FC > 1.5 threshold was applied to prioritize transcripts exhibiting robust and biologically meaningful changes in ac4C modification within our cellular context. Under this criterion, we identified four transcripts that displayed consistent alterations in ac4C enrichment—specifically, increased ac4C abundance in senescent cells and decreased ac4C levels upon NAT10 knockdown. Importantly, through multiple layers of downstream validation, including acRIP-qPCR, analysis of mRNA abundance, RNA stability assays, and protein-level validation, only DYRK1A consistently satisfied all validation criteria. These integrative analyses support DYRK1A as the most robust and functionally relevant NAT10-dependent ac4C target in senescent colonic epithelial cells.\n2. Fig. 3g needs quantification.\nWe have added quantitative results for Fig. 3g, including the fluorescence intensity of Ocln and the number of Ki67-positive epithelial cell. The corresponding quantitative data are presented in updated Supplementary Figure 3e and main text (Line150-151).\n3. The original data of Western blotting should be included.\nWe have provided western blotting data in “Source Data” file in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nThe authors satisfactorily addressed my comments.\nWe sincerely thank you for the careful evaluation of our revised manuscript and for the positive feedback.\nReviewer #4 (Remarks to the Author):\nThe authors have addressed most of my questions.\nA minor point remains regarding the reporter system (#8). They have added details about the construct, which is helpful.\nHowever, I would like to clarify my original question about“positive controls.” They included a CMV-driven luciferase (a technical control), but what I had in mind was a biological positive control, i.e., a condition known to induce DYRK1A transcription (if such a condition available). This is particularly important given that the conclusion is based on a negative result. Therefore, the insights from this data (ED Fig. 5e) in its current form are limited. Nevertheless, given the other supportive data (e.g., mRNA stability), I feel this could be a minor point.\nResponse:\nThank you for raising this point and we apologize for this confusion. As suggested, we should have built and validated a luciferase reporter for already known DYRK1A transcriptional regulator (such as REST in neuronal cells4, MEF2D in glioblastoma cells5) as a positive control that works in our cell context. We have clarified these interpretations in the discussion section to emphasize that incorporating appropriate biological positive controls would further strengthen the rigor of similar transcriptional reporter assays (Line 365-368). In the present study, the regulation of DYRK1A by RNA ac4C modification is supported by multiple evidences, including RNA decay assays, transcriptome-wide RNA sequencing following Actinomycin D treatment. We sincerely appreciate your insightful comments again.\nReferences\n1. Chen, C. et al. NAT10 Promotes Gastric Cancer Liver Metastasis by Modulation of M2 Macrophage Polarization and Metastatic Tumor Cell Hepatic Adhesion. Adv Sci (Weinh) 12, e2410263 (2025).\n2. Sun, L. et al. A critical role of N(4)-acetylation of cytidine in mRNA by NAT10 in T cell expansion and antiviral immunity. Nat Immunol 26, 619-634 (2025).\n3. Wang, J.N. et al. NAT10 exacerbates acute renal inflammation by enhancing N4-acetylcytidine modification of the CCL2/CXCL1 axis. Proc Natl Acad Sci U S A 122, e2418409122 (2025).\n4. Kim, M.Y. et al. A repressor complex, AP4 transcription factor and geminin, negatively regulates expression of target genes in nonneuronal cells. Proc Natl Acad Sci U S A 103, 13074-13079 (2006).\n5. Wang, P., Wang, L., Chen, L. & Sun, X. Dual-specificity tyrosine-phosphorylation regulated kinase 1A Gene Transcription is regulated by Myocyte Enhancer Factor 2D. Sci Rep 7, 7240 (2017)."}]

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