RNA cytidine acetyltransferase NAT10 maintains T cell pathogenicity in inflammatory bowel disease

Introducion

Inflammatory bowel disease (IBD) arises from a sophisticated interaction of genetic, environmental, and immunological elements, impacting an approximated 7 million patients worldwide¹. The imbalance in T cell immune responses significantly promotes the progression of IBD, underscoring the need for a more profound investigation into the molecular mechanisms that regulate T cell behavior as the disease evolves. In recent years, the role of RNA modifications in epigenetic regulation has garnered significant attention. There are over 170 types of RNA modifications constituting the epitranscriptome². Research has found that the epitranscriptome expands the regulatory functions of RNA by affecting various aspects of RNA metabolism, thereby opening up new avenues for gene regulation^2,3,4. Previous investigations have underscored the importance of RNA modifications in regulating T cell immunity^{5,6,7,8,9,10,11}. Nonetheless, the majority of these studies have concentrated on the realm of RNA methylation, leaving a void in our knowledge regarding other forms of RNA modification. Among these, N⁴-Acetylcytidine (ac⁴C), unique as the only known acetylation process in RNA, has emerged as a highly conserved modification in eukaryotic RNA2,^2,12,13, playing a pivotal role in diverse cellular processes. However, the involvement of ac⁴C in the development and function of immune cells is still unclear.

N-acetyltransferase 10 (Nat10), originally identified as a lysine acetyltransferase (KAT), is now universally recognized as the exclusive enzyme responsible for the acetylation of ac⁴C on mRNA^12,14,15. NAT10-mediated ac⁴C acetylation has been observed to regulate various post-transcriptional processes, including enhancing mRNA stability and translation¹², maintaining tRNA conformation^15,16, and aiding in ribosome maturation^14,17, thus regulating human embryonic stem cell (hESC) fate transitions¹⁸, tumorigenesis^19,20, and human accelerated aging syndrome^21,22. Nevertheless, the role of ac⁴C in modulating T cell homeostasis and its pathogenic effects in the context of IBD is yet to be identified.

Here, we found that Nat10 levels are associated with the activation of inflammatory and apoptotic pathways within CD4⁺ T cells in patients with IBD. To further investigate its role, we generated mice with lineage-specific deletion of NAT10 in T cells and found that Nat10-deficient mice exhibited a pronounced decline in both proportion and number of CD4⁺ and CD8⁺ T cells under steady state in peripheral immune organs compared to wild-type (WT) mice. Besides, Nat10-deficient naïve CD4⁺ T cells failed to induce adoptive transfer colitis. After analyzing the RNA-seq data, we found a significant enrichment of genes related to the apoptotic pathway in Nat10-deficient CD4⁺ T cells. Subsequent experiments demonstrated that Nat10 could directly bind to the mRNA encoding the anti-apoptotic factor BCL2-associated athanogene 3 (Bag3), thereby maintaining its stability. The absence of NAT10 elicits the downregulation of the Bag3 gene, setting off a series of downstream events that include the upregulation of genes associated with apoptosis and an escalated apoptotic rate of T cells. Our research unveils a previously unknown function of the NAT10-ac⁴C-Bag3 axis in preserving the homeostatic balance of T cells.

Results

Nat10 expression correlates with inflammatory and apoptotic pathways in human ulcerative colitis CD4⁺ T cells

IBD, encompassing Crohn’s disease and ulcerative colitis (UC), is characterized by chronic inflammation of the gastrointestinal tract, with T cells playing a pivotal role in the pathogenesis. To explore the role of NAT10-mediated ac⁴C acetylation in regulating T cell activity during IBD, we analyzed previously published single-cell transcriptomes of sorted T cells isolated from inflamed and non-inflamed colonic tissues of patients with UC, and colonic T cells from healthy subjects and patients with UC in remission as control groups²³. The T cells were effectively clustered into CD4⁺ and CD8⁺ populations (Fig. 1a). Our analysis revealed that, in comparison with samples from healthy tissues, Nat10 expression was diminished in CD8⁺ T cells from non-inflamed colonic tissue and remained largely unchanged in CD8⁺ T cells from inflamed tissues (Fig. 1b). However, in CD4⁺ T cells, Nat10 expression was notably elevated in non-inflamed colonic tissues and exhibited a substantial increase in inflamed tissues (Fig. 1b). These results suggest that NAT10-mediated ac⁴C modification in CD4⁺ T cells may play a role in the progression of UC. We further explored the single-cell sequencing (scRNA-seq) data and found that in CD4⁺ T cells lacking Nat10 expression, there was a significant enrichment of highly expressed genes associated with apoptosis-related signaling pathways (Fig. 1c), implying that Nat10 expression in CD4⁺ T cells links to the regulation of apoptosis in human UC.

**Fig. 1: *Nat10* expression in CD4⁺ T cells links to inflammation and apoptosis signaling in human ulcerative colitis.**

Nat10 expression is selectively enhanced in response to T cell activation

Previous studies have highlighted the dynamic nature of RNA modification-related genes during T cell activation^5,6,7. However, these studies mainly focus on RNA methylation and largely remain unknown about other RNA modifications, especially the only RNA acetylation-ac⁴C that is catalyzed by Nat10. This has prompted us to delve deeper into the significance of Nat10’s role throughout this process. Therefore, we performed a systematic quantification of Nat10 expression during T cell activation^7,24, and observed a gradual increase in both protein and RNA expression at various time points post-activation (Fig. 2a, b). This initial observation was intriguing and suggests a potential correlation between Nat10 expression and the progression of T cell activation.

**Fig. 2: T cell-specific deletion of *Nat10* leads to disrupted splenic T cell homeostasis.**

To validate these findings and further explore the temporal dynamics of Nat10 expression, we conducted an in vitro experiment. We isolated naïve CD4⁺ T cells from peripheral lymphoid organs of WT mice. These cells, representing the initial, unactivated state of T cells, were then stimulated with anti-CD3/CD28 antibodies, which are known to mimic the natural activation signals that T cells receive during an immune response. The activation was carried out at several time points: 0, 3, 6, 24, 48, and 96 h, allowing us to capture the early and late stages of T cell activation. Following the activation, we purified the proteins from these time-point samples and performed western blot analysis to assess the protein expression levels of NAT10 (Fig. 2c). The results were consistent with our initial findings, showing that NAT10 expression starts from a basal level in the naïve state and then increases progressively over time. This expression pattern implies that Nat10 might play a role in modulating T cell function.

Nat10 is not essential for thymic T cell development

To further elucidate the physiological role of Nat10 in T cells, we conducted a detailed investigation by employing a conditional gene knockout strategy. This strategy was designed to selectively remove Nat10 in T cells, utilizing transgenic Cre recombinase that is driven by the regulatory regions of the mouse Cd4 gene. Upon successful generation of the Nat10^flox/flox Cd4^Cre (cKO) mice and WT littermates (Supplementary Fig. S1a), we performed quantitative reverse transcription PCR (RT-qPCR) and western blot analysis to assess the levels of Nat10 mRNA and protein respectively in CD4⁺ T cells, and found the expression of Nat10 was effectively ablated in CD4⁺ T cells isolated from cKO mice, indicating that our conditional knockout approach was successful (Supplementary Fig. S1b, c).

To explore the consequences of Nat10 deficiency on T cell development, we conducted a comparative analysis of T cell subsets in the thymus of WT and cKO mice. The thymus is the primary site where T cells mature and differentiate into various functional subsets. We examined the distribution and frequency of different T cell populations, including double-positive (DP) CD4⁺CD8⁺ and single-positive (SP) CD4⁺ or CD8⁺ T cells, as well as double-negative (DN) subsets, and observed that the absence of Nat10 did not lead to any significant disruption in the developmental stages of T cells within the thymus (Supplementary Fig. S1d–f). These observations suggest that the lack of Nat10 does not impede the latter stages of T cell maturation within the thymus.

Nat10 is crucial for preserving T cell homeostasis

Having established that Nat10 does not affect thymic T cell development, we shifted our focus to examine the role of Nat10 in the peripheral lymphatic system, which is crucial for T cell function and survival outside the thymic environment. We found that the proportion and number of total T cells were considerably decreased in the spleen and peripheral lymph nodes (mesenteric and inguinal lymph nodes) of cKO mice, compared to their WT counterpart (Fig. 2d–f and Supplementary Fig. S2). Further analysis revealed that the numbers of both CD4⁺ and CD8⁺ T cells were simultaneously reduced (Fig. 2d–f and Supplementary Fig. S2). We then assessed the activation status of T cells and found that both CD4⁺ and CD8⁺ T cells in the cKO mice were in a more activated state (Fig. 2d, g, i and Supplementary Fig. S2). However, due to the significant reduction in the absolute numbers of CD4⁺ and CD8⁺ T cells, the total number of cells at effector stages was decreased or unchanged (Fig. 2d–j). These findings indicate that the absence of Nat10 has a detrimental effect on the maintenance of T cell populations in the periphery.

In order to explore the cause of the reduced T cell number resulting from NAT10 deficiency, we examined the proliferation, apoptosis and migratory marker expression of T cells. Our findings indicated an increase in the proliferation (Ki67 expression) and migratory ability (CXCR3 expression) of CD4⁺ T cells when Nat10 was absent (Fig. 3a, b and Supplementary Fig. S3a–d). However, we also observed a significant increase in the apoptosis (Annexin V expression) of CD4⁺ cells following the knockout of Nat10 at steady state (Fig. 3a, b and Supplementary Fig. S3a–d). This enhanced apoptosis may be the principal reason for the decreased number of T cells in the absence of Nat10. Further analysis revealed that the absence of Nat10 led to an increased expression of cytokines (granzyme B and IFN-γ) (Fig. 3a, b and Supplementary Fig. S3a–d). In addition, we also detected the levels of Fas expression, a marker of activation-induced cell death, in CD4⁺ T cells under steady state and after anti-CD3/CD28 antibodies stimulation. We found that, regardless of the state, Nat10-deficient CD4⁺ T cells expressed higher levels of Fas compared to WT cells (Fig. 3c, d and Supplementary Fig. S3e, f). These results collectively indicates that the absence of Nat10 leads to a hyperactivated state of T cells.

**Fig. 3: *Nat10*-deficient naïve CD4⁺ T cells show hyperactivation and increased apoptosis.**

Nat10 regulates the ability of CD4⁺ T cells to initiate spontaneous colitis

Adoptive transfer colitis serves as a well-established T cell-based model of inflammatory bowel disease^25,26. We commenced by isolating naïve CD4⁺ T cells from both WT and Nat10 cKO mice, and then transferred them separately into Rag1^−/− recipient mice, thereby inducing an adoptive transfer colitis model. Throughout the course of the experiment, we monitored the body weight of the recipient mice every week as a measure of their health and response to the transferred T cells. We found that the mice that received the WT naïve CD4⁺ T cells exhibited a noticeable weight loss during the process (Fig. 4a). This weight loss is indicative of the onset of colitis, a common manifestation in this model. In contrast, the mice that received the Nat10-deficient naïve CD4⁺ T cells not only maintained their weight but also exhibited a continued weight gain (Fig. 4a). Furthermore, these mice displayed a longer colon length compared to those injected with WT naïve CD4⁺ T cells (Fig. 4b, c), suggesting a potentially altered disease progression in the absence of Nat10.

**Fig. 4: The absence of *Nat10* leads to an impaired ability of CD4⁺ T cells to induce adoptive transfer colitis.**

To gain deeper insights into the long-term effects of Nat10 deficiency on the transferred T cells, we analyzed the composition of donor CD4⁺ T cells 10 weeks post the induction of adoptive transfer colitis, and observed a significant difference in the persistence of these cells within the recipient mice. Specifically, the Nat10 KO CD4⁺ T cells were largely depleted from the spleen and peripheral lymph nodes of the recipients compared with WT cells (Fig. 4d, e and Supplementary Fig. S4), indicating a possible role of Nat10 in the survival and maintenance of naïve CD4⁺ T cells in vivo.

Further investigation into the underlying cause of this depletion revealed a pronounced increase in the apoptosis (Annexin V expression) of CD4⁺ T cells derived from Nat10 cKO mice when compared to those from WT mice (Fig. 4d, e). This is also consistent with the increased apoptosis of Nat10 KO cells under homeostasis (Fig. 3a, b). The heightened apoptosis in the absence of Nat10 could be a critical factor contributing to the diminished presence of these cells in the lymphatic organs, thus reducing their capacity to induce adoptive transfer colitis. These findings imply the importance of Nat10 in regulating T cell homeostasis and suggest its potential as a target for therapeutic intervention in immune-related disorders.

Nat10 deficiency caused the enrichment of genes in apoptosis-related pathways

Given the phenotype observed in Nat10 cKO mice, which includes disrupted T cell homeostasis and heightened apoptosis, we proceeded to explore the molecular underpinnings of these effects. We isolated naïve CD4⁺ T cells from WT and Nat10 cKO mice for RNA-seq, and then established a fold-change threshold of greater than 2 and a p < 0.05 to identify genes that were differentially expressed. Our analysis unveiled a substantial shift in gene expression, with hundreds of genes exhibiting either increased or decreased expression levels in the Nat10-deficient naïve CD4⁺ T cells (Fig. 5a). Intriguingly, the predominantly upregulated genes were found to be linked to immune response mechanisms and the process of apoptosis (Fig. 5b), a pattern that aligns with the observations from human IBD samples (Fig. 1c). Conversely, the genes that were downregulated were notably enriched in biological pathways related to visual perception (Fig. 5c), suggesting a broader impact of Nat10 deficiency on cellular functions beyond the immune system. To validate the RNA-seq findings, we conducted qPCR and western blot experiments and confirmed that the expression levels of genes known to promote apoptosis, such as Bax and Dapk2, were significantly elevated in Nat10 KO naïve CD4⁺ T cells (Fig. 5d, e).

**Fig. 5: *Nat10* deficiency results in an upregulation of apoptosis-related genes in naïve CD4⁺ T cells.**

Considering the established role of Nat10 in histone acetylation¹⁸, which is a key epigenetic modification influencing gene expression, we hypothesized that Nat10 might also modulate chromatin accessibility. To test this, we performed assays for transposase-accessible chromatin using sequencing (ATAC-seq). However, we found that the genes proximal to regions with altered chromatin accessibility post-Nat10 KO did not significantly overlap with the differentially expressed genes identified by RNA-seq. This observation suggests that Nat10’s influence on gene expression may not be mediated through changes in chromatin accessibility (Supplementary Fig. S5).

To ascertain whether the observed reduction in the number of Nat10-deficient naïve CD4⁺ T cells was indeed a consequence of increased apoptosis, we employed an in vitro treatment with Z-VAD-FMK, a potent and pan-caspase inhibitor²⁷. Our results demonstrated that the heightened apoptosis observed in Nat10-deficient naïve CD4⁺ T cells could be effectively mitigated by this treatment (Fig. 5f, g). Thus, the deletion of Nat10 leads to the upregulation of apoptosis-related genes of naïve CD4⁺ T cells without alterations in chromatin accessibility, indicating that Nat10’s role in regulating apoptosis may be independent of its effects on the epigenome.

The ablation of Nat10 results in reduced Bag3 mRNA stability

Extensive research has established that Nat10’s role on mRNA molecules is primarily to augment their stability and enhance their translational efficiency^12,14,15. It follows that the genes that were upregulated in Nat10 KO naïve CD4⁺ T cells are unlikely to be direct targets of Nat10. To elucidate how Nat10 modulates T cell apoptosis, our investigation pivoted to the downregulated genes, seeking those that might be directly influenced by Nat10. We embarked on a comparative analysis, overlapping our RNA-seq data with previous ac⁴C-seq results¹², which led us to identify a potential anti-apoptotic target: Bag1 and Bag3 (Fig. 6a). Both Bag1 and Bag3 are recognized for their anti-apoptotic properties, and they possess ac⁴C modification sites on their mRNAs. However, by using qPCR and western blot, we confirmed that while Bag1 mRNA and protein expression remained largely unchanged in Nat10-deficient CD4⁺ T cells, Bag3 expression was notably diminished (Figs. 5d and 6b).

**Fig. 6: Characterization of NAT10’s role in stabilizing *Bag3* mRNA.**

To substantiate that Bag3 is a direct target of Nat10, we conducted RNA immunoprecipitation (RIP) experiments. These experiments demonstrated that Bag3 mRNA could be significantly enriched through the immunoprecipitation of the NAT10 protein (Fig. 6c), providing evidence of a direct interaction between NAT10 protein and Bag3 mRNA. Further, to decipher the mechanism by which Nat10 regulates Bag3 expression, we conducted RNA decay experiments. In isolated naïve CD4⁺ T cells, the transcriptional arrest induced by Actinomycin D allowed us to observe that the degradation rate of Bag3 mRNA in Nat10 KO CD4⁺ T cells was markedly faster compared to that in WT cells (Fig. 6d). This finding suggests that Nat10 promotes the stability of Bag3 mRNA, thereby curbing excessive apoptosis in naïve CD4⁺ T cells and ultimately maintaining T cell homeostasis and pathogenicity (Supplementary Fig. S6).

Discussion

The immune system functions as a tightly regulated network, relying on precise gene expression control for effective operation. T cells, a vital component of the adaptive immune response, maintain immune homeostasis through a delicate balance of proliferation, differentiation, and apoptosis. Any disruption in their development or functional execution can significantly impact immune surveillance. In this work, our findings reveal that NAT10 deficiency causes a notable disturbance in T cell development under steady-state conditions and mitigates T cell pathogenicity during IBD. This finding is pivotal as it underscores NAT10’s previously unrecognized role in sustaining T cell homeostasis and functionality through RNA ac⁴C modification.

Our previous research, in conjunction with others, has established that RNA modifications such as m¹A and m⁶A are crucial for maintaining T cell homeostasis and pathogenicity^{5,6,7,8,9,10,11}. However, these studies have primarily focused on RNA methylation, resulting in a significant gap in knowledge regarding alternative RNA modification mechanisms. NAT10, with its unique role as the sole known enzyme for ac⁴C modification, has garnered substantial interest. This enzyme is implicated in various aspects of RNA metabolism, yet its specific function in preserving T cell functionality has remained elusive and controversial. A recent study observed a significant reduction in the RNA expression of NAT10 in CD4⁺ T cells from systemic lupus erythematosus (SLE) patients compared to healthy controls, along with a decrease in ac⁴C modification²⁸. This suggests that NAT10-mediated ac⁴C modification may suppress the onset and progression of SLE. In contrast, our bioinformatics analysis of single-cell data from inflamed and non-inflamed colonic tissues of IBD patients showed that higher NAT10 expression in CD4⁺ T cells correlates with increased inflammatory activity, indicating a potential role for NAT10 in promoting IBD progression. These different observations highlight the complexity of ac⁴C in regulating T cell function.

While previous work has shown a potential correlation between NAT10 expression in CD4⁺ T cells and SLE onset, it lacked direct in vivo evidence²⁸. Our study illuminates the essential role of NAT10 in maintaining in vivo T cell homeostasis and its influence on the pathogenicity of CD4⁺ T cells in inducing IBD. Specifically, we observed that the deletion of Nat10 leads to significant disruptions in T cell homeostasis, characterized by heightened cytokine secretion, increased proliferation, and elevated apoptosis, suggesting a hyperactivated state in T cells, aligning with a recent study that indicates mice with T cell-specific NAT10 deficiency display reduced numbers of mature T cells in peripheral lymphoid organs²⁹. However, their study emphasizes NAT10’s role in T cell activation and proliferation through acetylating RACK1 to affect cellular metabolism, while our study focuses on NAT10’s function in epitranscriptomics, with a particular focus on the ac⁴C modification of RNA related to apoptosis regulation. Mechanistically, our RNA-seq analysis revealed that differentially expressed genes were predominantly involved in apoptosis-related pathways, with the anti-apoptotic gene Bag3 playing a key role in NAT10-mediated T cell survival. The downregulation of Bag3 in the absence of NAT10 triggers a cascade leading to heightened apoptosis. Bag3, known to inhibit cell death by interacting with anti-apoptotic proteins such as BCL2 and HSP70, is a well-established regulator of apoptosis^{30,31,32,33,34,35,36,37}. Our research indicates that NAT10’s effect on Bag3 mRNA stability is critical for controlling T cell survival.

In conclusion, our study provides new insights into the role of NAT10 as an RNA cytidine acetyltransferase in safeguarding T cell balance through the NAT10-ac⁴C-Bag3 axis, offering a novel perspective on post-transcriptional regulation in inflammatory diseases. This has profound implications for advancing our understanding of T cell biology and immune regulation. Further studies will be necessary to understand the molecular mechanisms underlying these effects fully and to explore the potential therapeutic implications of modulating NAT10 activity in T cell-related inflammatory diseases. As our exploration into NAT10’s immune system function is still in its infancy, additional studies are crucial for a complete understanding of its extensive impact on immune processes and its potential application in therapeutic strategies.

Materials and methods

Mice

Nat10^flox/flox mice were genetically engineered using the CRISPR/Cas9 system, which involved the insertion of two loxP sites flanking the exon 4. Cd4^Cre mice were procured from the Jackson Laboratory, Nat10 ^flox/flox mice were crossed with Cd4^Cre mice to produce Nat10 ^flox/flox Cd4^Cre offspring lacking NAT10 expression in CD4⁺ T cells, with Nat10 ^flox/flox mice serving as WT controls. All mice were raised and maintained in a specific pathogen-free environment at Cyagen Co., Ltd. Both male and female mice aged 8–12 weeks were utilized in experiments. Animals were randomly assigned to experimental groups, with each cage containing mice from different groups. The Institutional Animal Care and Use Committee (IACUC) of Shanghai Jiao Tong University School of Medicine approved all animal procedures (No. JUMC2023-086-A).

Cell isolation

The thymus, spleen, and lymphoid node were pressed through a fine mesh of 200-gauge. To obtain splenic single-cell leukocyte suspensions, the erythrocytes were lysed using a red cell lysis buffer from ThermoFisher Scientific.

Adoptive transfer colitis model

Naïve CD4⁺ T cells were isolated from spleens utilizing the EasySep Mouse Naïve CD4⁺ T Cell Isolation Kit (STEMCELL Technologies). These cells were subsequently labeled with CellTrace Violet (ThermoFisher Scientific). Subsequently, 5 × 10⁵ naïve CD4⁺ T cells from Nat10 ^flox/flox Cd4^Cre mice and their WT littermate controls were transferred into Rag1^–/– mice, respectively. Body weight measurements of the recipient mice were taken weekly.

T cell isolation and stimulation

Naïve CD4⁺ T cells were purified using the EasySep Mouse Naïve CD4⁺ T Cell Isolation Kit (STEMCELL Technologies) following the provided instructions. Subsequently, the purified T cells were stimulated in replicate wells of a 96-well plate using plate-bound anti-CD3 (2 μg/mL) and anti-CD28 (2 μg/mL) antibodies, and cultured in RPMI with 10% fetal bovine serum (TransGen Biotech), 1% streptomycin/penicillin, β-mercaptoethanol (40 nM) and IL-2 (2 ng/mL, ThermoFisher Scientific) for different time points.

Antibody staining and flow cytometry

Monoclonal antibodies (mAbs) specific to CD3 (clone 145-2C11), CD4 (clones RM4-5, RM4-4, GK1.5), CD8 (clone 53-6.7), CD25 (clone 3C7), CD44 (clone IM7), CD62L (clone MEL-14), CD45 (clone 30-F11), CXCR3 (clone CXCR3-173), IFN-γ (clone XMG1.2), Granzyme B (clone, Granzyme B), Ki67 (clone 16A8), and TCR-β (clone H57-597) were purchased from BioLegend. The Fas antibody (clone Jo2) was purchased from BD Biosciences. The cells were incubated with the antibodies (diluted 1:200) at 4 °C for 30 min to facilitate staining. For intracellular cytokine staining, the cells were first stimulated with a combination of phorbol myristate acetate (50 ng/mL; Sigma), ionomycin (1 μg/mL; Sigma), and GolgiPlug (1 μL/mL; BD Biosciences) for a duration of 4 h. Following surface staining with antibodies, the cells were fixed and permeabilized using a Fixation/Permeabilization Solution Kit from BD Biosciences. Subsequently, the intracellular molecules were stained with specific antibodies. For intracellular transcription factor staining, the cells were fixed and permeabilized using the FOXP3/Transcription Factor Buffer Set provided by BioLegend. The resulting data were collected using a BD Fortessa X20 flow cytometer (BD Biosciences) and analyzed using FlowJo software.

Apoptosis assay

The PE or APC Annexin V Apoptosis Detection Kit, along with 7-AAD (BioLegend), was utilized to assess the apoptotic status of the cells.

RNA library preparation, sequencing, and differentially expressed genes analysis

Naïve CD4⁺ T cells from Nat10^flox/flox Cd4^Cre and Nat10^flox/flox mice were isolated using the EasySep Mouse CD4⁺ T Cell Isolation Kit. Total RNA was extracted by the TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RNA purity and quantification were assessed by NanoDrop 2000 spectrophotometer (ThermoFisher Scientific, USA), and the integrity was evaluated by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA libraries were prepared with NEBNext^® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB) according to the manufacturer’s instructions.

The libraries were sequenced on Illumina HiSeq X Ten platform (150 bp paired-end reads) by Shanghai Neo-Biotechnology Co., Ltd (Shanghai, China). About 50 million raw reads for each sample were generated. After processing raw reads by Trimmomatic, the high-quality clean reads were mapped to the mouse genome (GRCm38.p6) using HISAT2³⁸. FPKM³⁹ of each gene was calculated by Cufflinks ⁴⁰, and the read counts of each gene were obtained by HTSeq-count⁴¹. The DESeq2⁴² was used to normalize the raw counts and identify differentially expressed genes (DEGs; |fold change| ≥ 2; p < 0.05). Volcano plots and bar plots were performed using the R package. KEGG pathway enrichment analysis of DEGs was performed using the phyper function from R package “stats” based on the hypergeometric distribution. Two independent biological replicates were performed for RNA Sequencing.

RT-qPCR

Naïve CD4⁺ T cells were subjected to Total RNA isolation using the TRIzol reagent (Invitrogen), strictly adhering to the manufacturer’s guidelines. Subsequently, the isolated RNA was reverse-transcribed utilizing the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme). For real-time PCR, ChamQ Universal SYBR qPCR Master Mix (Vazyme) was employed. Additionally, Actb served as an internal control for the quantification of relative mRNA amounts. The primer sequences utilized for qPCR are enumerated as follows: Actb (forward, 5′-GCCAACCGTGAAAAGATGAC-3′; reverse, 5′-CATCACAATGCCTGTGGTAC-3′), Bax (forward, 5′-TGAAGACAGGGGCCTTTTTG-3′; reverse, 5′-AATTCGCCGGAGACACTCG-3′), Dapk2 (forward, 5′-CTCGATGAGGAGCCCAA-ATATG-3′; reverse, 5′-AATTCGCCGGAGACACTCG-3′), Cdk1 (forward, 5′-AGAAGGTACTT-ACGGTGTGGT-3′; reverse, 5′-GAGAGATTTCCCGAATTGCAGT-3′), Il12b (forward, 5′-TGGT-TTGCCATCGTTTTGCTG-3′; reverse, 5′-ACAGGTGAGGTTCACTGTTTCT-3′), Il17a (forward, 5′-TTTAACTCCCTTGGCGCAAAA-3′; reverse, 5′-CTTTCCCTCCGCATTGACAC-3′), Il18 (forward, 5′-GACTCTTGCGTCAACTTCAAGG-3′; reverse, 5′-CAGGCTGTCTTTTGTCAACG-A-3′), Ifng (forward, 5′-ATGAACGCTACACACTGCATC-3′; reverse, 5′-CCATCCTTTTGCCAG-TTCCTC-3′), Tnfsf4 (forward, 5′-AATCTGGAAAACGGATCAAGGC-3′; reverse, 5′-CAGGCA-GACATAGATGAAGCAC-3′), Bag3 (forward, 5′-CTGGGAGATCAAAATCGACCC-3′; reverse, 5’-GCTGAAGATGCAGTGTCCTTAG-3′), Bag1 (forward, 5′’-GGAGGAAATGGAAACACCC-3′; reverse, 5′-AGTCTTGGACAACTGGCTCAC-3′), Gapdh (forward, 5′-GAGAGTGTTTCCTCGT-CCCG-3′; reverse, 5′-GCCTCACCCCATTTGATGTTAG-3′).

RNA degradation assay

RNA degradation assay was performed following previously established protocols ⁶. Naïve CD4⁺ T cells were isolated using the EasySep Mouse CD4⁺ T Cell Isolation Kit and seeded into a 96-well plate with a cell density of 5 × 10⁵ cells per well. Actinomycin-D (Sigma-Aldrich) was then added to each well, achieving a final concentration of 5 μM. Subsequently, CD4⁺ T cells were collected at 2 and 4 h post-addition of actinomycin-D. The harvested cells were processed according to the protocol outlined in the “RT-qPCR” section, and the resulting data were normalized to the t = 0 time point for comparison.

Western blot

Naïve CD4⁺ T cells from spleen and lymphoid nodes were purified by EasySep Mouse CD4⁺ T Cell Isolation Kit (Stemcell). The total protein from CD4⁺ T cells was extracted by RIPA lysis buffer (Beyotime, P0013B) supplemented with protease inhibitors (Roche, 11697498001). The primary antibodies used were as follows: NAT10 (Abcam, ab194297, 1:1000); BAX (Cell Signaling Technology, 2772, 1:1000); DAPK2 (Abclonal, A8199, 1:1000); IFN-γ (Abclonal, A12450, 1:1000); BAG1 (Abcam, ab32109, 1:500); BAG3 (Abcam, ab92309, 1:1000). They were diluted in 5% no-fat milk buffer and incubated at 4 °C overnight. After washing the membrane with 0.1% TBST buffer three times, the HRP-conjugated secondary antibody was added to the membrane and incubated at room temperature for 1 h. The final signal was detected by SuperFemto ECL Chemiluminescence Kit (Vazyme) using ChemiDoc MP Imaging System (Biorad), and ACTB (Cell Signaling Technology, 3700, 1:5000) was used as the internal control.

ATAC-seq

For each sample, 30,000 naïve CD4⁺ T cells from WT and Nat10 cKO mice with 90% viability were spun down for cell pellet collection. After being resuspended in nuclear lysis buffer, the pellets were collected and treated with the TruePrepTM DNA Library Prep Kit V2 (Vazyme, TD501) following the manufacturer’s instructions. PCR was used to amplify the transposed DNA fragments using primers with different Barcodes. Illumina Hiseq X ten (PE150) was used to sequence the library. Bowtie 2 (version 2.2.3) was used to map the data to the Mus Musculus (mm10) reference genome, and MACS2 (version 2.1.1) was used to identify binding sites with the parameter ‘-q 0.05–nomodel–extsize 150–keep-dup all–call-summits’.

RIP-qPCR

Naïve CD4⁺ T cells were lysed in polysome lysis buffer (PLB, 0.5% NP40, 100 mM KCl, 5 mM MgCl₂, 10 mM HEPES, 1 mM DTT, 100 units/mL RNase inhibitor, and 1× protease inhibitor cocktail, pH7.0) by mechanical homogenizer and centrifuged at 20,000× g for 20 min at 4 °C to remove the debris. The supernatant was pre-cleared with blocked Dynabeads and immunoprecipitated with antibody-conjugated (Proteintech, 13365-1-AP) or normal IgG-conjugated Dynabeads at 4 °C for at least 3 h. Next, the beads were washed four times with NT2 buffer (0.05% NP40, 50 mM Tris-HCl, 150 mM NaCl, and 1 mM MgCl₂, pH 7.4) supplemented with RNase and proteinase inhibitor. Before the last washing, the beads were divided into two portions. A small portion was used to isolate protein for enrichment identification of target protein, the remaining portion was resuspended in 100 μL of NT2 buffer supplemented with RNase inhibitor and 30 μg of proteinase K to release the RNA at 55 °C for 30 min, and the RNA was eluted using 1 mL of TRIzol.

Statistical analysis

To compare pairs of groups, we employed unpaired Student’s t-test and two-way analysis of variance (ANOVA). All the collected data are represented as the mean ± standard error of the mean (SEM). Statistically significant differences were determined when the values of p < 0.05.