Fig. 5: Hypomethylation of m1G9 in mt-Leu(UAA) in cybrids.
a Left: A primer extension analysis of WT and the A3243G variant of mt-Leu(UAA), using cells validated for MRPP1-KD (Fig. S5e). Neg controls are from cybrids transfected with a scrambled siRNA. The m1G9 level was the fraction of the RT stop at position 10 in the sum of positions 10, 8, and the primer. Right: Relative m1G9 levels to the negative control in bar graphs (blue for WT; red for A3243G). Data are the average ± SD (n = 3). b A Northern analysis of the stability of mt-Leu(UAA), showing size markers superimposed from a stained gel. c Data in (b) in a bar graph, where the steady-state level of mt-Leu(UAA) relative to 5S rRNA was quantified for each and normalized to that of WT in the negative control and d compared to that of the WT in the same targeting condition. Data (c, d) are the average ± SD (n = 3). e Steady-state levels of τm5U34 in a CMC-assisted primer extension analysis of WT and A3243G, showing the fraction of A35 in the sum of A35, τm5U34, U33, and C32 and reported as the level in the CMC (+) lane subtracted from that of the corresponding CMC (−) lane. f Data in (e) in a bar graph, where the τm5U34 level in mt-Leu(UAA) was quantified for each and normalized to that of WT in the negative control and g compared to that of the WT in the same targeting condition. Data (f, g) are the average ± SD (n = 4). h Steady-state levels of charged mt-Leu(UAA) in an acid-urea gel. i Data in (h) in a bar graph, where the steady-state level of mt-Leu(UAA) was quantified for each and normalized to that of WT in the negative control and j compared to that of the WT in the same targeting condition. Data (i, j) are the average ± SD (n = 4). Source data are provided as a Source Data file, and p values were from two-sided paired t test (a, c, d, f, g, i, j).
