The knockdowns of m6A methylome components affect PAN RNA expression levels. The normalized mRNA expression (y-axis) of (A) RBM15, METTL3, and FTO in BCBL-1 knockdowns (KD) cell lines. The x-axis corresponds to uninduced (0 h pi) and lytic (8–72 h pi) stages of KSHV infection. Control samples represent latent BCBL-1 cells treated with scrambled siRNAs. (B) Western blots representing the efficiency of knockdowns on the level of protein expression. Tubulin was used as a control. (C) PAN RNA expression in knockdown cell lines versus control at specified time points of KSHV replication. (D) ORF50 (left) and ORF57 (right) mRNA expression levels in knockdown versus wild-type BCBL-1 cells (control) at indicated time points post-induction. MALAT1 and β-actin were used for data normalization as their expression levels were not significantly affected by the knockdowns and progression of KSHV infectivity cycle (n = 4). (E) The representative native PAGE indicating frequency of modification at m6A sites in BCBL-1 RMB15, METTL3, and FTO knockdown (KD) cells. L represents 50 bp DNA ladder. The analyzed time points of infection (0 h pi, 48 h pi), negative (−4Sed) and positive (+4Sed) reactions are indicated on top. The position of products specific for modified and unmodified sites is indicated to the left. The column graphs represent the average modification frequency for each site at 0 h pi and 48 h pi time point of infection in specified knockdown cell lines. Standard deviations for frequency measurements are indicated.
