TRMT5 methylates mitochondrial (mt) and cytosolic (cyto) tRNAs at position 37. (A-B) Illustration of mt-tRNAs methylated by TRMT5 at position G37 (A) and quantification of m¹G levels in mt-tRNAs using mass spectrometry (B). Modification levels are normalized to N6-threonyl-carbamoylation, a universal modification of adenosine 37 in tRNAs. (C-D). Integrative genomics viewer snapshot of nanopore sequenced mt-tRNA^Pro(TGG) (C) and mt-tRNA^Ala(TGC) (D) from OCI-AML2 Ctr (scr; top) and TRMT5- KO (sg2) cells. Nucleotides with mismatch frequencies >0.05 are in color (panels C-D) and in gray otherwise. (E) Principal component analysis of summed error values from mitochondrial (left) and cyto (right) tRNAs in OCI-AML2 Ctr and KO cells. N = 3 infections per conditions. (F-G) Relative summed error of all potential TRMT5 targets (G37) in KO cells compared with Ctr in OCI-AML2 (F) and Kasumi-1 (G) cells. (H) Alpha-fold model predicting TRMT5 protein structure. Green highlights the predicted mitochondrial transfer peptide (left; 1) and the TRM (right; 2). (I) Western blot detecting TRMT5 protein levels in Ctr and rescued TRMT5 KO cells. Cells were infected with scr or sg2. KO cells were rescued by reexpressing TRMT5 WT, catalytic dead (mutant [MUT]) or lacking the MTS constructs. Ctr cells (sg2 and scr) were rescued with an empty vector (EV) Ctr. Vinculin (VCL) serves as a loading Ctr. Dotted line indicates different exposure times of the same blot. (J) FC of relative summed error in TRMT5 mt (n = 3) and (cyto; 12) tRNA targets in the indicated rescued TRMT5 KO lines. Mean ± standard deviation (panels F-G). Mean ± standard error (panel J). Two-tailed paired t test (panel J). TRM, tRNA methylation motif.