PUS1 regulates mitochondrial translation through downregulation of specific mitochondrial tRNAs. (A) Heat map of the amount of mt-tRNAs differentially expressed in MLASA-iPSCs and MLASA-Res-iPSCs. P < .05 and fold change (FC) >1.2; n = 3. (B) Simplified secondary structures of 5 downregulated mt-tRNAs in MLASA-iPSCs. Potential Ψ sites that may be modified by PUS1 are marked in red. The 5' and 3' ends of the tRNAs are labeled. The yellow arrows indicate the targeted regions of the designed primers for the CMC primer extension assay. (C) Primers specific for mt-tRNA^Cys (left), mt-tRNA^Ser(UCN) (middle), or mt-tRNA^Tyr (right) were used in primer extension reactions to determine the location of Ψ in MLASA and MLASA-Res iPSCs as described in “Methods.” The reverse transcription stops, corresponding to residue Ψ28, are labeled with red triangles. Full length indicated, the fragment from the beginning of the primer to the 5’ end of the tRNA. Primer, the unbound primers. (D) Ranking according to the sum of usage frequency of codons complementary to mt-tRNA^Cys, mt-tRNA^Ser(UCN), and mt-tRNA^Tyr in each mitochondrial-encoded protein. (E-F) Western blot analyses (E) and densitometry (F) of the mitochondrial-encoded proteins examined in iPSCs. Protein levels are normalized to β-actin or β-tubulin. n = 2. (G-H) Western blot analyses (G) and densitometry (H) of the nuclear-encoded oxidative respiratory chain proteins examined in iPSCs. Protein levels are normalized to β-actin or β-tubulin (n = 2). (I) qRT-PCR analyses for mRNA expression levels of some selected mitochondrial-encoded and nuclear–encoded oxidative respiratory chain genes in iPSCs. Expression levels are normalized to 18S (n = 3). Values in all panels denote mean ± SD (∗P < .05; ∗∗P < .01; ∗∗∗P < .001); unpaired Student t test.