Analyzing 12S rRNA Methylation in GTPBP6-Deficient Cells

To ensure that the accumulation of NSUN4 on the mtLSU in GTPBP6-deficient cells does not affect its second function as a methyltransferase modifying the 12S rRNA at position 1488, we analyzed the modification status of 12S-m⁵C1488 and, as a control, 12S-m⁴C1486, by subjecting DNase-treated total RNA from HEK293T wild-type and Gtpbp6^−/− cells to bisulfite sequencing^{50 (link),51 (link)}. Bisulfite treatment was performed using the EpiTect Bisulfite kit (Qiagen) according to the manufacturer’s instructions. Deamination was performed by three cycles of incubation at 70 °C for 5 min and at 60 °C for 60 min. Samples were purified using mini Quick spin RNA columns (Roche) and the desulphonated in Tris pH 9.0 for 30 min at 37 °C. RNA was extracted using phenol:chloroform, precipitated and reverse transcribed from the 12S-m⁵C841_RT primer (Primer sequences: Supplementary Table 4) using Superscript III reverse transcriptase (Thermo) according to the manufacturer’s instructions. A 70-nt fragment of the 12S rRNA was amplified by PCR (Primer sequences: Supplementary Table 4) and cloned using a TOPO-TA kit (Thermo). Clones were sequenced at Eurofins Genomics using the T7 primer and only sequences in which all cytosines (disregarding C1486 and C1488) were converted to uracil/thymine were used for the presented analysis (Supplementary Fig. 6c).