Figure 2: Base-resolved mapping of ac4C in HeLa mRNA.
(A) RedaC:T-seq protocol and pipeline for determination of ac4C sites through NaBH4-induced C:T mismatches.
(B) Distribution of C:T mismatches in 18S rRNA from wildtype (WT, red) and NAT10−/− (dark blue) HeLa. Positions 1337 and 1842 correspond to known ac4C sites.
(C) Frequency of all mismatch types observed in RedaC:T-seq prior to filtering relative to untreated or NAT10−/− NaBH4-treated controls
(D) Volcano plot depicting the change in mismatch ratio vs. significance comparing wildtype and NAT10−/− HeLa RedaC:T-seq results. Color coding indicates significant differences (FDR adj p≤0.05, Fisher’s exact test, ≥5 fold).
(E) Cumulative frequency plot of C:T mismatch frequency distribution in wildtype and NAT10−/− RedaC:T-seq reads.
(F) Median local RedaC:T-seq coverage at all detected ac4C sites in wildtype HeLa as compared to untreated control.
(G) Browser views of example start codon proximal RedaC:T-seq defined ac4C sites showing: (top) wildtype HeLa acRIP-seq density (Arango et al., 2018), (middle) wildtype and NAT10−/− HeLa RedaC:T-seq, and (bottom) total and poly(A) HeLa ac4C-seq (Sas-Chen et al., 2020).
(H) Sequencing depth at RedaC:T-seq defined ac4C sites in the current study and previous HeLa ac4C-seq.
(I) Metagene plot of transcript coverage in HeLa RedaC:T-seq (red) and reanalysis of published HeLa ac4C-seq (purple). Solid lines show all mRNAs while dashed lines reflect coverage for RedaC:T-defined acetylated mRNAs.