Fig 2: UBASH3A inhibits phosphorylation of NF-κB dependent on TCR signaling. A, B CD4+ T cells were transfected with pcDNA3.1-UBASH3A and empty vector. A At 24 h after transfection, UBASH3A mRNA levels were quantified by RT-qPCR. The amount of UBASH3A transcript was expressed relative to that of the GAPDH transcript. B At 30 min after TCR stimulation, Western blotting was performed with a series of antibodies. Data are representative of three independent Western blotting experiments. C The amount of total (t)-NF-κB was normalized to that of β-actin and that of phosphorylated (p)-NF-κB was then normalized to that of normalized t-NF-κB. Results were plotted on a histogram. Values are expressed as mean ± standard deviation. *P<0.05, vs lane 1 or 4, by Dunnett’s multiple comparison test. RQ, relative quantification

Fig 3: Downregulation of UBASH3A in CD4+ T cells of rheumatoid arthritis (RA) patients. A In silico screening for RA-susceptibility factors in the super-enhancer database (dbSUPER), GWAS, and biogene portal system (BioGPS). B Super-enhancer (SE_17452) domain mapped to the UBASH3A locus in CD4+ T cells. The locus is mapped to chromosome 21 at approximately 4,382,000–4,388,000. Histone H3 containing the acetylated lysine 27 (H3K27ac) marks are provided in GM12878, which shows lymphoblastoma cell line. Rs1893592-A/C is located in the intron between exons 10 and 11, marked with an asterisk. C The mRNA expression levels of the UBASH3A gene in CD4− and CD4+ T cells of peripheral blood mononuclear cells (PBMCs) collected from healthy donors (HD, n=11) and RA patients (n=24) were evaluated by RT-qPCR. D Expression levels of UBASH3A mRNA among three allele groups of patients with RA (rs1893592-AA, AC, and CC). E Expression levels of UBASH3A protein in CD4− and CD4+ T cells of HD (n=3) and RA patients (n=3) evaluated by Western blotting. β-actin was used as a loading control. C, D Amount of UBASH3A transcript expressed relative to that of GAPDH transcript. Data are mean ± standard deviation. Dunnett’s multiple comparison test. F UBASH3A protein expression in CD4+ T cells from the lymph nodes of dermatomyositis (DM) and RA patients evaluated by immunofluorescence staining. RQ, relative quantification. *P<0.05, by Student’s t test and ANOVA. Scale bar = 100 μm

Fig 4: UBASH3A inhibits the production of IL-6 induced by TCR signaling in CD4+ T cells. A–D CD4+ T cells collected from healthy donors (HD) (A, B) and rheumatoid arthritis (RA) patients (C, D) were transfected with mock (lanes 1, 5, 9, and 13) and pcDNA3.1-UBASH3A (lanes 2, 6, 10, and 14) without CD3/CD28 stimulation for 24h, or mock (lanes 3, 7, 11, and 15) and pcDNA3.1-UBASH3A (lanes 4, 8, 12, and 16) with CD3/CD28 stimulation for 24h. After that, proinflammatory cytokine mRNA levels (A and C) were measured by RT-qPCR, and cytokine levels (B and D) were measured by cytometric bead array. E The relative mRNA expression levels of UBASH3A and IL-6 were analyzed in CD4+ T cells of the isolated PBMCs from patients with RA (n=24). Spearman’s test was used for the correlation analysis between two variables of interest. The amount of cytokines transcript was expressed relative to that of GAPDH transcript. Data are mean ± standard deviation from three independent experiments. *P<0.05, vs mock or mock+CD3/CD28, by Dunnett’s multiple comparison test. RQ, relative quantification

Fig 5: eRNA promotes UBASH3A transcription in CD4+ T cells. A Genomic structure of UBASH3A, histone H3 containing the acetylated lysine27 (H3K27ac), and transcripts peaks. Putative eRNA_1-3 are expressed within the super-enhancer (SE_17452) in CD4+ T cells. H3K27ac are marked in GM12878 lymphoblastoid cells. RNA-sequencing data are shown in GM12878 and K562 leukemia cells. B Each antisense LNA GapmeR. LNA-eRNA denotes LNA needed for eRNA knockdown. C, D Jurkat cells were transfected with LNA-NC (lanes 1, 4, and 7), LNA-eRNA1_S (lane 2), LNA-eRNA1_AS (lane 3), LNA-eRNA2_S (lane 5), LNA-eRNA2_AS (lane 6), LNA-eRNA3_S (lane 8), and LNA-eRNA3_AS (lane 9). S and AS denote sense and antisense, respectively. The amount of each transcript was quantified by RT-qPCR and then expressed relative to that of GAPDH transcript. Data are mean ± standard deviation of three independent experiments. *P<0.05, vs control, by Dunnett’s multiple comparison test. RQ, relative quantification