Fig 1: In vitro assay for the processing of mitochondrial tRNACys precursors. (A, B) tRNACys precursors for 5′ or 3′ end processing assays. Thirty-six nucleotides (nt) of 5′ and 3′ end leaders of tRNACys were shown, including the m.5783C > T substitution. (C) In vitro 5′ end processing assays. Processing assays with mitochondrial RNase P were carried out in parallel for wild type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™ to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. (D) Relative processing efficiencies of tRNACys precursors catalyzed by RNase P. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. (E) In vitro 3′ end processing assays. Processing assays with mitochondrial RNase Z were undertaken in parallel for wild-type and mutant substrates. Samples were withdrawn and stopped after 5, 10, 15, 20 and 25 min, respectively. Reaction products were resolved by denaturing polyacrylamide gel electrophoresis and reacted with a chemiluminescent substrate CDP-Star™ to detect the chemiluminescent. The graph shows the results of a representative experiment reaction. (F) Quantification of the efficiencies of tRNACys precursors catalyzed by RNase Z. The relative processing efficiencies were calculated from the initial phase of the reaction. The calculations were based on three independent determinations. (G) Western blot analysis of tRNA processing related proteins MRPP2, ELAC2 and TRNT1 with β-Actin as a loading control. (H) Quantification of MRPP2, ELAC2 and TRNT1. Average relative values of MRPP2, ELAC2 and TRNT1 were normalized to the average values of β-Actin in various cell lines. The values for the latter are expressed as percentages of the average values for the control cell lines. The average of three independent determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 2.