The RAP MS analysis for PAN RNA m6A methylome and effect on PAN m6A methylation frequencies. (A) Venn diagrams representing the overlap between results generated from formaldehyde (FA) and UV cross-linked PAN-protein complexes. The three distinct biological replicates are indicated with colors (I-III). (B) Principal component analysis (PCA) of proteins identified in RAP MS (UV, x-axis) and FA experiments (y-axis). Proteins involved in regulation of the PAN m6A status are marked with purple dots, and they include the writer complex recruiter RBM15, readers HNRNPC, YTHDF2, SND1, and the FTO eraser. (C) Normalized expression of PAN RNA assessed by RT qPCR in non-cross-linked total RNA samples extracted during KSHV latent (0 h pi) and lytic (48 h pi) stages of replication, and after the PAN RNA–protein cross-linking and affinity capture. (D) Results of western blot analyses performed on whole cell BCBL-1 lysates (left) and captured PAN RNA-protein complexes (right) during uninduced (0 h pi) and lytic (8–72 h pi) stages of KSHV replication. β-actin was used as a control.
