Fig. 4

EBNA1 promotes ADAR1 translation but does not affect its mRNA stability. a Integrative Genomics Viewer (IGV) tracks displaying the distribution of m⁶A peaks and EBNA1 binding peaks in the ADAR1 transcript. b MeRIP experiments were performed using an anti-m⁶A antibody, followed by qPCR analyses in HK1 or HONE1 cells. Rabbit IgG served as a control. The enrichment of the indicated genes was normalized to the input level. c MeRIP experiments were performed in C666-1 cells using an anti-m⁶A antibody, followed by qPCR analysis. Rabbit IgG was used as a control. The enrichment of the indicated genes was normalized to the input level. d FLAG(EBNA1)-RIP-qPCR and IGF2BP3-RIP-qPCR were performed in HONE1 and HK1 cells (OE EBNA1 group and NC group) to analyze the effect on ADAR1 mRNA enrichment. e In C666-1 cells, EBNA1 was knocked down, and EBNA1-RIP-qPCR and IGF2BP3-RIP-qPCR were performed to analyze their effects on ADAR1 mRNA enrichment. f After EBNA1 overexpression, Western blotting was used to examine ADAR1 protein levels in NPC cells (HK1, HONE1). g After IGF2BP3 was knocked down in NPC cells (HK1, HONE1), ADAR1 protein levels were detected. h In EBNA1-overexpressing HEK293FT cells, IGF2BP3 was knocked down followed by reintroduction of IGF2BP3-MUT1 or IGF2BP3-WT, and changes in ADAR1 protein levels were examined. i An RNA pull-down assay was used to confirm the enrichment of the IGF2BP3 protein on ADAR1 mRNA. j Co-IP and RNA pull-down assays were performed, followed by mass spectrometry, to identify proteins interacting with EBNA1, IGF2BP3, and ADAR1 mRNA, respectively. Venn analysis was conducted to analyze the three groups of proteins. k An EBNA1-FLAG plasmid was transfected into HEK293FT cells, and Co-IP was performed using anti-FLAG and anti-EIF4G1 antibodies to isolate the EBNA1-FLAG and EIF4G1 complexes, respectively. Western blotting was used to detect the presence of FLAG (EBNA1) and EIF4G1. l IF staining was performed using anti-IGF2BP3, anti-FLAG, and anti-EIF4G1 antibodies to detect the colocalization of exogenous FLAG (EBNA1), endogenous IGF2BP3, and EIF4G1 proteins in EBNA1-overexpressing HK1 cells. Cellular nuclei were stained with DAPI (blue). EIF4G1, FLAG (EBNA1), and IGF2BP3 were visualized in red, pink, and green, respectively. The signals of the two proteins and three proteins were merged, respectively. Scale bar: 50 µm. m Sucrose gradient-based polysome profiling analyses were conducted on NC and OE EBNA1 HK1 cells. n ADAR1 mRNA in each polysome fraction was quantified via qRT-PCR and plotted as a percentage of the total amount. o Sucrose gradient-based polysome profiling analyses were performed on control cells and EBNA1-knockdown cells in C666-1. p qRT‒PCR was used to quantify ADAR1 mRNA in each polysome fraction of shNC and shEBNA1 C666-1 cells, and the results were plotted as a percentage of the total amount. q Knockdown of EIF4G1 in OE EBNA1 HK1 and NC HK1 cells, followed by Western blot analysis of ADAR1 protein levels. b–q The results are representative of three independent experiments. b–e The data are shown as the mean ± s.e.m. Two-way ANOVA with Tukey’s test was used for multiple comparisons. **P < 0.01; ***P < 0.001