FIG 15.

KSHV lytic switch protein RTA (ORF50) induces m⁶A and enhances its own pre-mRNA splicing. (A) Western blot detection of RTA and β-tubulin in iSLK-BAC16 cells without (Control) and with doxycycline stimulation for 24 h and 293T cells at 48 h posttransfection with an equal amount (4 μg) of pRTA-3×FLAG (pRTA) or the empty vector. (B) Dot blot detection of m⁶A in 10 μg total RNAs isolated from the cells described in the legend to panel A, using an antibody to m⁶A and subsequent chemiluminescence detection. The blot was also stained with ethidium bromide (EtBr). (C) Relative levels of total mRNA and m⁶A-mRNA of ORF50 (RTA) in iSLK-BAC16 cells treated as described in the legend to panel A. (D) ORF50 (RTA) mRNA-to-pre-mRNA ratios in 293T cells cotransfected with wild-type pExon1-intron-exon2-GFP (WT; 2 μg) plus pRTA-3×FLAG (pRTA; 2 μg) or the empty vector (Vector; 2 μg) or mutant plasmids with site A or F mutated (Mut-A and Mut-F, respectively) with similar cotransfection combinations. *, differences that were statistically significant with P values of <0.05.