Fig 2: CRISPR/Cas9 system-mediated NSUN2 and NSUN6 depleted in HEK293T. (A) Schematic diagram of plasmid construction of PX459 for knockdown of NSUN2 and NSUN6 genes using CRISPR/Cas9 technology. (B) Schematic representation the construction of NSUN2−/− and NSUN6−/− Cell line. (C) Agarose gel electrophoresis results of PCR products to verify the knockdown of NSUN2 and NSUN6. M represents the DNA Marker DL2000 used. (D) Schematic representation of knockout targeting the human methyltransferase NSUN2 and NSUN6 genes. NSUN2 and NSUN6 exons are indicated by columns, primers F and R were used to assay knockout efficiency, and the sequence information below shows the number of missing bases. (E, G) Schematic representation of the amino acids encoded by the wild-type, NSUN2−/− (E) and NSUN6−/− (G) HEK293T cell lines, with changes in the encoded synthesized proteins after base deletions. (F, H) The knockout efficiency of NSUN2 (F) and NSUN6 (H) in HEK293T cell lines verified by Western blotting. The protein level of GAPDH was served as loading controls.

Fig 3: DRAM detection system was assayed in an 5-methylcytosine (m5C)-dependent form.(A, B) Two m5C sites from RPSA (A) and AP5Z1 (B) mRNA detected by deep sequencing of bisulfite-sequencing PCR in HEK293T cells. The m5C sites are highlighted by red color. The m5C fraction of RPSA and AP5Z1 were 75.5% and 27.25% (the number of reads is greater than 1000). (C, D) Sanger sequencing following RT-PCR verified two m5C sites from RPSA (C) and AP5Z1 (D) mRNAs in DRAM-transfected HEK293T cells, respectively. HEK293T cells only expressing DRAMmut or Deaminase were served as negative controls. The left panel illustrates the location of DRAM-induced mutation sites, which is highlighted in red asterisk. The right panel shows the corresponding quantification of Sanger sequencing (n=3). (E, F) The knockout efficiency of NSUN2 (E) and NSUN6 (F) in HEK293T cell lines verified by western blotting. The protein level of α-Tubulin and GAPDH were served as loading controls, separately. (G, H) DRAM-induced mutations close to m5C sites in AP5Z1 (G) and RPSA (H) mRNAs after NSUN2 and NSUN6 knockout in HEK293T cells. The left panel illustrates the location of DRAM-induced mutation sites, which is highlighted in red asterisk. The right panel shows the corresponding quantification of Sanger sequencing (n=3). Figure 2—source data 1.PDF file containing the original western blots for Figure 2E and F, labeled with relevant bands and processing methods. Figure 2—source data 2.Original western blots for Figure 2E and F.

Fig 4: DRAM detection system was assayed in an 5-methylcytosine (m5C)-dependent form.(A, B) Two m5C sites from RPSA (A) and AP5Z1 (B) mRNA detected by deep sequencing of bisulfite-sequencing PCR in HEK293T cells. The m5C sites are highlighted by red color. The m5C fraction of RPSA and AP5Z1 were 75.5% and 27.25% (the number of reads is greater than 1000). (C, D) Sanger sequencing following RT-PCR verified two m5C sites from RPSA (C) and AP5Z1 (D) mRNAs in DRAM-transfected HEK293T cells, respectively. HEK293T cells only expressing DRAMmut or Deaminase were served as negative controls. The left panel illustrates the location of DRAM-induced mutation sites, which is highlighted in red asterisk. The right panel shows the corresponding quantification of Sanger sequencing (n=3). (E, F) The knockout efficiency of NSUN2 (E) and NSUN6 (F) in HEK293T cell lines verified by western blotting. The protein level of α-Tubulin and GAPDH were served as loading controls, separately. (G, H) DRAM-induced mutations close to m5C sites in AP5Z1 (G) and RPSA (H) mRNAs after NSUN2 and NSUN6 knockout in HEK293T cells. The left panel illustrates the location of DRAM-induced mutation sites, which is highlighted in red asterisk. The right panel shows the corresponding quantification of Sanger sequencing (n=3). Figure 2—source data 1.PDF file containing the original western blots for Figure 2E and F, labeled with relevant bands and processing methods. Figure 2—source data 2.Original western blots for Figure 2E and F.