Fig 1: Summary of mitochondrial tRNA-metabolic processes revealed in the study. Schematic diagram summarizing the main results. Mitochondrial tRNAs are denoted by their decoding specificity. CCA denotes normal 3' termini, CCAAAA denotes oligoadenylated tRNA and CC followed by the longer A run denotes polyadenylated tRNA. Square brackets indicate that some polyadenylated tRNAs are also truncated at CCA, to a degree that is tRNA-specific. Oligoadenylation is also induced by EtBr in some tRNAs (Lys and His indicated as examples, although differing in their susceptibility), and involving an unknown enzyme. PDE12 is required for rapid removal of oligo(A) from these tRNAs, as reported previously (21). EtBr treatment or the A3243G mutation introduce a structural distortion of the relevant tRNA (indicated by a kink in the structure), resulting in deacylation and adenylation. tRNA polyadenylation in response to EtBr treatment requires mtPAP and SUV3, while turnover of polyadenylated tRNAs (dashed cloverleaf) requires PNPase and SUV3. The accumulation of the polyadenylated A3243G mutant tRNA is stimulated by doxycycline (DOX), which inhibits both tRNA polyadenylation and turnover in cells treated with EtBr. Dashed arrows denote minor or uncertain pathways, including low-level polyadenylation of the A3243G mutant tRNALeu(UUR) in cells not treated with doxycycline.
Fig 2: Enzymes required for mitochondrial tRNA polyadenylation and turnover. Northern blots probed for mitochondrial tRNASer(UCN), following EtBr treatment in 143B wild-type cybrid cells also treated with cordycepin (CDY) or siRNAs against different genes as indicated. (A) Cells pre-treated for 1 h and then continuously throughout the experiment with or without 20 µg/ml cordycepin, as shown. (B) Cells pre-treated for 96 h with siRNAs as indicated (see Supplementary Table S1), or mock-transfected, before 6 h of treatment with or without EtBr, as shown. (C) Cells pre-treated for 72 h with siRNAs as indicated (see Supplementary Table S1), or untreated, before 6 h of treatment with or without EtBr, followed by recovery for the indicated times after removal of the drug. In each siRNA experiment shown, knockdown at the protein level was verified by western blots, representative examples of which are shown in Supplementary Figure S4B and D. 72 h or 96 h of knockdown gave essentially identical results, both on Western and Northern blots. Signals detected by phosphorimaging in parts (B, C). Note that we confirmed the effects of knockdown of mtPAP, SUV3 and PNPase using alternative siRNAs (Supplementary Figure S4E, F, G).
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