Extended Data Fig. 10: Ψ sites in mt-tRNA and mitochondrial targets assigned to PUS1.

(a) Schematic diagram of PRAISE to identify Ψ sites in mitochondrial tRNAs. Total small RNAs are first treated by Alkb demethylase to demethylase m¹A and m³C, and then treated with 85% sulfite/15% bisulfite solution. RNA was then followed by 3’ adapter ligation, reverse transcription, 5’ linker ligation, PCR, and sequencing. The ‘Untreated’ sample is set as a negative control. (b) Venn diagram depicting the overlap of Ψ sites detected in mitochondrial tRNA between PRAISE and previously reported method (Suzuki T et al.⁴¹). (c) Deletion rates of 51 known Ψ sites and 1 novel of Ψ sites in mitochondrial tRNA. For each site, deletion rates of treated and untreated are marked with different symbols. (d) Sequence alignment and comparation of PUS1 isoform 1 and isoform 2 in human cell line. Mitochondrial target sequence of PUS1 isoform 1 is marked in red color. (e) Double immunofluorescence staining of HeLa cells transfected with expression vectors encoding Flag-tagged PUS1 isoform 1 and 2 (green) and DAPI (blue). The left panels show the expression vectors, and the right panels show the green and blue images merged in Adobe Photoshop. Representative images were obtained under the same exposure condition from n = 3 biological repeats. (f) Deletion rates of Ψ sites in mitochondrial RNA (mt-RNA) in PUS1 KO and HEK293T cells. The horizontal axis represents deletion rate of mt-RNA Ψ sites in HEK293T cells, and the vertical axis represents deletion rate of mt-RNA Ψ sites in PUS1 KO cells. (g) Sequencing depth of regions surrounding Ψ3286 in mt-Leu tRNA and the corresponding deletion rate in HEK293T (WT) and TRUB1 KO treated samples are plotted. (h) Deletion rate of Ψ sites in mt-RNA in PUS7 KO and HEK293T cells. The horizontal axis represents deletion rate of mt-RNA Ψ sites in HEK293T cells, and the vertical axis represents deletion rate of mt-RNA Ψ sites in PUS7 KO cells.