Fig. 3

Validation and NSUN2-dependence of candidate m⁵C sites in mRNA and ncRNA. Amplicon-specific bsRNA-seq was performed with total RNA isolated from HeLa cells after siRNA-mediated knockdown targeting NSUN2 or TRDMT1 along with control siRNAs (targeting DNMT1 or a non-targeting control [NTC]; see Fig. S5 for knockdown efficiency; see Table S6 for read coverage details). Grids showing cytosine position along the analysed transcript section (in columns; genomic coordinates for the first and last cytosine, and the candidate m⁵C site are given; knockdown sample indicated on the left) with each square coloured by observed cytosine non-conversion (white-to-red colour scale; shown in panel b). The longest transcript isoform (based on Ensembl) is shown above the grid with candidate m⁵C site position (red circle) and sequence context indicated (non-converted cytosine in red). The enzyme identified to mediate methylation is also indicated: N – NSUN2; ? – unknown. ‘Confirmatory’ data (N = 1) using NSUN2, TRDMT1, DNMT1 and NTC samples was generated for sites with high coverage. ‘In-depth’ data (N = 2 to 3) using NSUN2, TRDMT1 and NTC samples was obtained for lowly covered sites. Asterisks indicate Student’s t test p value between a given sample and the NSUN2 knockdown. See Fig. S6 for additional candidates. a ‘Confirmatory’ data (left panel) for mRNA candidate sites in RPS3 (ribosomal protein S3), NDUFB7 (NADH:ubiquinone oxidoreductase subunit B7), RTN3 (reticulon 3), SZRD1 (SUZ RNA binding domain containing 1) and OSBPL8 (oxysterol binding protein-like 8). ‘In-depth’ data (right panel) shows the average for sites in RPS3, GIPC1 (GIPC PDZ domain family member 1), SRRT (serrate) and GID8 (GID complex subunit 8 homologue). b ‘In-depth’ data for two mRNA candidate sites (PIGG [phosphatidylinositol glycan anchor biosynthesis class G] and ZDHHC8 [zinc finger DHHC-type containing 8]) that showed no response to either NSUN2 or TRDMT1 knockdown. c ‘Confirmatory’ data for ncRNA candidate sites in CAGE1 (cancer Antigen 1), NSUN5P2 (NSUN5 pseudogene 2), SNORD62B (small nucleolar RNA, C/D box 62B), RPPH1 (ribonuclease P RNA component H1) and SCARNA2 (small Cajal body-specific RNA 2)