FIG 9.

Specific m⁶A sites in ORF50 (RTA) pre-mRNA are responsible for splicing. (A) m⁶A sites in the ORF50 (RTA) locus determined by MeRIP-seq. (B) Genomic locations of m⁶A consensus GGAC sites in the ORF50 (RTA) locus of the KSHV genome and presentation of the pExon1-intron-exon2-GFP plasmid. (C) ORF50 (RTA) mRNA-to-pre-mRNA ratios in 293T cells transfected with equal amounts (4 μg) of wild-type (WT) pExon1-intron-exon2-GFP or its mutants with mutations at each of the individual m⁶A sites (Mut-A to Mut-J) (GGAC → GGCC). (D) Western blot detection at 48 h posttransfection of RTA and β-tubulin in the 293T cells described in the legend to panel C. (E) Representative images of GFP expression in the cells described in the legend to panel C. The statistical significance of the differences in the ORF50 (RTA) mRNA-to-pre-mRNA ratio between cells transfected with the wild-type plasmid and cells transfected with a plasmid harboring any mutant was analyzed by an unpaired t test. *, differences with P values of <0.05 (n = 3).