Fig. 4: NAT10 is required for the stability and translation efficiency of Myc mRNA.
a, Volcano plot showing differentially expressed genes between CKO and FLOX activated TEFF cells, with cutoffs of P ≤ 0.05 and | fold change | ≥ 2; FC, fold change. b, Volcano plot showing differentially acetylated peaks between CKO and FLOX TEFF cells, with cutoffs of P ≤ 0.00001 and | fold change | ≥ 2. c, Metagene analysis of ac4C sites identified on mRNAs from FLOX and CKO TEFF cells; UTR, untranslated region; CDS, coding sequence. d, Venn diagram showing the numbers of transcriptionally repressed transcripts with significantly fewer modified by ac4C. e, KEGG pathway analysis of the overlapped genes identified in d. f, Cumulative distribution function plot exhibiting differential expression of ac4C+ or ac4C– transcripts between CKO and FLOX TEFF cells. g, Volcano plots of differentially expressed mRNAs between CKO and FLOX TEFF cells segregated by ac4C modification, with cutoffs of P ≤ 0.05 and | fold change | ≥ 2. h, GSEA of ‘MYC targets V1’ between CKO and FLOX TEFF cells; NES, normalized enrichment score. i, Circular heat map showing mRNA levels of MYC-regulated genes in FLOX and CKO TEFF cells. j, MYC, CDC25A, CDK2 and CDK4 protein levels in FLOX and CKO CD3+ TN cells stimulated with anti-CD3/CD28 for the indicated lengths of time. Representative bands of three independent experiments are presented. k, IGV plot displaying specific ac4C peaks on Myc transcripts. Peaks are represented as subtracted read densities (IP minus input). l, ac4C RIP–qPCR for Myc mRNA in activated FLOX and CKO CD3+T cells; n = 3; **P = 0.0017. m, Degradation of Myc mRNA in activated FLOX and CKO CD3+ T cells 1 and 2 h after actinomycin D treatment; n = 3; ***P = 0.0003 and **P = 0.0097. n, NAT10 RIP–qPCR for Myc mRNA in activated CKO and FLOX CD3+ T cells; n = 4; ****P < 0.0001. o, Ribosome occupancy of Myc and control mRNAs in activated CKO and FLOX CD3+ T cells; n = 3; ****P < 0.0001. n refers to biologically independent samples. Activated T cells were stimulated with anti-CD3/CD28 for 24 h in l–o. Error bars represent mean ± s.e.m. (l–o). Data were analyzed by two-tailed, negative binomial distribution test (a, b and g); one-sided, hypergeometric test (Benjamini–Hochberg adjusted) (e); two-tailed, Mann–Whitney U-test (f); two-tailed Kolmogorov–Smirnov test (h) and two-tailed, unpaired t-test (l–o).
