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. 2022 Sep 26;50(18):10695–10716. doi: 10.1093/nar/gkac817

Figure 4.

Figure 4.

Human NOP2/NSUN1 methylates the 28S rRNA at position C4447. (A) HCT116 cells were transfected with non-targeting control (siC), NOP2/NSUN1 siRNA#1 or siRNA#2. 72h later, NOP2/NSUN1 depletion efficiency was determined by western blot. (B) A fraction of cells from (A) were harvested for nuclear RNA extraction. Nuclear RNA was treated with bisulfite salt and the region surrounding C4447 was amplified by RT-PCR for sequencing on the Illumina Mi-Seq platform. Non-converted (protected by 5mC modification) and converted cytosines (lacking m5C modification) near C4447 were plotted. The converted cytosines at position 4447 are highlighted by yellow bars. (C) Percentage of converted cytosine (C) at position 4447 on 28S rRNA. (D) Percentage of converted cytosine on vtRNA1.2. Total RNA from HCT116 cells was treated with bisulfite salt and vtRNA1.2 was amplified by RT-PCR for sequencing on the Illumina Mi-Seq platform. The data are shown as the mean of three independent biological replicates ± standard deviation (SD). Statistical significance between NOP2/NSUN1 depleted samples and non-targeting siRNA control samples was calculated using a two-tailed independent Student's t-test.