Fig 1: NAT10 promotes B cell activation, and IgA production through noncanonical NF-κB signaling.(A) Proliferation assays of splenic B cells were conducted in vitro, either without stimulation (NT) or with the specified inducers. These assays measured the proliferation of splenic B cells in response to the indicated stimuli (n = 5). (B) Intracellular IgA levels in splenic B cells were analyzed after 5 days of culture with aIgM (10 μg/ml) alone or combined with TGF-β (2 ng/ml), anti-CD40 (α-CD40; 1 μg/ml), or BAFF (200 ng/ml). The percentage of IgA+ B cells was quantified by flow cytometry, with numbers shown in the outlined regions (n = 4). (C) ELISA was used to measure IgA levels in the supernatants of splenic B cells cultured for 5 days with aIgM alone or together with TGF-β, anti-CD40, or BAFF (n = 4). (D) qPCR was performed to assess α-GLT and AID mRNA expression in splenic B cells after 5 days of culture under the same conditions as in (B). The results are presented as fold changes relative to Actb mRNA levels and normalized using Bio-Rad CFX Manager 3.1 (n = 4). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences (A) was determined by t test, and those (B–D) were determined using one-way ANOVA with Newman–Keuls post-hoc test. **P < 0.01; ***P < 0.005. Source data are available online for this figure.
Fig 2: NAT10 inhibitor, Remodelin, enhances IgA production via noncanonical NF-κB pathway.(A) Proliferation assays of splenic B cells were incubated with complete medium containing 20 µM Remodelin, and stimulated with the specified inducers. These assays measured the proliferation of splenic B cells in response to the indicated stimuli (n = 5). (B) Intracellular IgA levels in splenic B cells were incubated with complete medium containing 20 µM Remodelin, and were analyzed after 5 days of culture with αIgM (10 μg/ml) alone or combined with TGF-β (2 ng/ml), anti-CD40 (α-CD40; 1 μg/ml), or BAFF (200 ng/ml). The percentage of IgA+ B cells was quantified by flow cytometry, with numbers shown in the outlined regions (n = 4). (C) qPCR was performed to assess α-GLT and AID mRNA expression in splenic B cells after 5 days of culture under the same conditions as in (B). The results are presented as fold changes relative to Actb mRNA levels and normalized using Bio-Rad CFX Manager 3.1 (n = 4). (D) ELISA was used to measure IgA levels in the supernatants of splenic B cells cultured for 5 days of culture under the same conditions as in (B) (n = 4). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences (A) was determined by t test, and those (B, C) were determined using one-way ANOVA with Newman–Keuls post-hoc test. **P < 0.01; ***P < 0.005. Source data are available online for this figure.
Fig 3: NAT10-mediated acetylation of NIK mRNA regulates noncanonical NF-κB pathway and IgA+ B cell differentiation.(A) qPCR analysis performed on RNA extracted from spleen B cells derived from WT and NAT10cKO mice, stimulated with either anti-CD40 (α-CD40; 1 μg/ml) or BAFF (200 ng/ml) (n = 4). (B) qPCR analysis of Map3k14 and Gfp mRNA in HEK293T cells, transfected with various combinations of expression vectors for hemagglutinin-tagged human NIK (HA-hNIK)-GFP and Flag-tagged NAT10 WT or the NAT10-G641E mutant (n = 4). (C) IB analysis of specific proteins in HEK293T cells transfected with different plasmid constructs. (D) mRNA stability assays in WT and NAT10-deficient B cells pretreated with BAFF (200 ng/ml) for 24 h, followed by the addition of 3 μg/ml CHX to inhibit transcription. Cells were harvested at the indicated times post-CHX treatment, and the levels of NIK mRNA were quantified by qPCR (n = 5). Results are expressed as fold changes relative to Actb mRNA, normalized using Bio-Rad CFX Manager 3.1. (E) RNA immunoprecipitation (RIP) was conducted using an anti-NAT10 antibody, followed by qPCR to detect RNA enrichment in NAT10−/− B cells overexpressing either NAT10 WT or NAT10-G641E, and stimulated with BAFF (200 ng/ml) for 24 h (n = 4). (F) Sequence alignment of ac4C modification sites in NIK mRNA targeted by NAT10. Canonical bases are highlighted in red, and mutated ones in blue. (G, H) qPCR (G) and IB (H) analyses of Map3k14 and Gfp mRNA levels in HEK293T cells transfected with various plasmid combinations (n = 4). (I) mRNA stability assays in NIK−/− B cells infected with different plasmids expressing the specified proteins. These B cells were pretreated with BAFF (200 ng/ml) for 24 h before the addition of 3 μg/ml CHX to stop transcription. Cells were harvested at the indicated times post-CHX treatment, and NIK mRNA levels were assessed by qPCR (n = 4). Data are shown as fold changes relative to Actb mRNA, normalized using Bio-Rad CFX Manager 3.1. (J) Proportion of IgA+ cells among purified NIK−/− B cells overexpressing the indicated genes, cultured with different combinations of IgA-inducing factors, and analyzed for IgA class switching in vitro by flow cytometry using intracellular IgA and GFP staining (n = 4). All data are representative of biological replicates at least three independent experiments. Data are represented as the means ± SDs. The significance of differences (E) was determined by t test, and those (B, D, G, I, J) were determined using one-way ANOVA with Newman–Keuls post-hoc test. **P < 0.01; ***P < 0.005. Source data are available online for this figure.
Fig 4: NAT10-mediated regulation of IgA in B cells and its association with IBD pathogenesis.(A) ELISA was used to quantify IgA levels in fecal samples from healthy donors and newly diagnosed patients with Crohn’s disease (CD) or ulcerative colitis (UC) (n = 30). (B) The proportion of IgA+ B cells within the total CD19+ B cell population from colonic tissues of healthy donors and newly diagnosed CD or UC patients (n = 10) was assessed using flow cytometry. The percentages of cells in each gated region are indicated. (C) LC–MS/MS was employed to determine the relative ac4C/C content in total RNA extracted from IgA+ and IgA- B cells from colonic tissues of healthy volunteers (n = 10). (D) Immunoblotting (IB) was used to measure NAT10 protein levels in IgA+ and IgA- B cells from the colonic tissues of healthy volunteers (n = 3). (E) CD19+ B cells isolated from healthy volunteers were stimulated with BAFF (200 ng/ml) for the indicated times, and Nat10 mRNA levels were analyzed by RT-qPCR (n = 5). The data are presented as fold changes relative to ACTB mRNA levels, normalized using Bio-Rad CFX Manager 3.1. (F) Following BAFF stimulation for the indicated times, total cellular extracts from CD19+ B cells isolated from healthy volunteers were subjected to immunoblotting to detect NAT10 protein. (G) NAT10 expression was analyzed by qPCR in total CD19+ B cells isolated from the colonic tissues of healthy donors and newly diagnosed CD or UC patients (n = 10). Results are shown as fold changes relative to Actb mRNA levels, normalized with Bio-Rad CFX Manager 3.1. (H) NAT10 protein levels were assessed by immunoblotting in total CD19+ B cells from colonic tissues of healthy donors and newly diagnosed CD or UC patients (n = 3). (I) A scatterplot showing the linear regression correlation between NAT10 mRNA levels in B cells from the colonic tissues of newly diagnosed CD patients (n = 10) and their disease activity index (CDAI). All data are representative of biological replicates in three independent experiments. Data are represented as the means ± SDs. The significance of differences (C) was determined by t test, and those (A, B, E, G) were determined using one-way ANOVA with Newman–Keuls post-hoc test. **P < 0.01; ***P < 0.005. Source data are available online for this figure.
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