Figure 1 ELAC2 mutations and gene/protein structure. (a) Gene structure of ELAC2 with known protein domains (as defined in Saoura et al., 2017) of the gene product and localization of amino acid residues and splice sites (variants indicated in blue) affected by mutations. Intronic regions are not drawn to scale. (b) Conservation of human ELAC2 amino acid residues affected by mutations across Pongo tapanuliensis (Pt), Bos Taurus (Bt), Gallus gallus (Gg) and Danio rerio (Dr). For complete sequence alignment including Saccharomyces cerevisiae Trz1 used for structure modeling, see Figure S3

Figure 2 Kinetic parameters of the pathogenic ELAC2 variants. (a) Kinetic parameters of the previously reported ELAC2 variants (Haack et al., 2013) with mt‐tRNALeu(UUR) substrate. The graph shows k cat/K M value relative to WT (e.g., the quotient [k cat mutant]/[k cat WT]). The bars indicate standard error. *, ** above the bars indicate p‐values with a significance of 0.05 and 0.01, respectively, calculated using unpaired t‐test. (b) Kinetic parameters of the novel ELAC2 variants with mt‐tRNALeu(UUR) substrate analysed as per (A). (c) Kinetic parameters of selected novel ELAC2 variants with mt‐tRNAIle substrate analysed as per (A)

Figure 3 Analysis of unprocessed mitochondrial tRNA–mRNA intermediates. (a) Linear genetic map of mtDNA (numbering according to RefSeq accession number J01415) indicating mt‐rRNA (green), mt‐mRNA (olive) and mt‐tRNA (black). Noncoding sequences in white. The mt‐tRNAVal‐16S rRNA and mt‐tRNAMet‐ND2 mRNA junctions are indicated by red brackets. LSP – Light strand promoter. HSP – Heavy strand promoter. (b) Northern blot processing analysis of the mt‐tRNAVal‐16S rRNA junction in total RNA samples of control fibroblasts (C1‐C3), fibroblasts from the previously published cases (57415, 61982, 65937; Haack et al., 2013) and fibroblasts from the patients harboring novel ELAC2 mutations (P1–4, P6–9, and P12). (c) Northern blot analysis of the processing of the mt‐tRNAMet‐ND2 mRNA junction. Samples as per (b). Asterisks indicate partially degraded RNA samples that were reanalyzed in a different blot and presented in the same panel

Figure 4 ELAC2 substitutions mapped on the structure of Saccharomyces cerevisiae RNase Z. The structure of S. cerevisiae RNase Z (Trz1, PDB#5MTZ; Ma et al., 2017) is shown in cartoon using PyMol. The amino domain, inter‐domain linker, and carboxy domain are colored green, blue, and pale green, respectively. Three views are shown to effectively visualize all the substitutions. All 13 ELAC2 substitutions (3 published previously (Haack et al., 2013), and 10 novel) are shown in all three views. Residues are labeled with S. cerevisiae RNase Z numbers and the numbers in brackets are for the H. sapiens ELAC2 residues. Some residues are not conserved between S. cerevisiae and H. sapiens RNase Z. Residues Arg68, Phe154, Gln388 localized in the amino domain are marked in red; Leu422 and Leu423 are in linker (also marked in red); Pro493, Thr520, Arg564, Lys660, Arg680, Tyr729, His749, Arg781 are in the carboxy domain and indicated in purple. (a) View with amino domain up, carboxy domain down and linker behind with the H. sapiens residues Phe154, Leu422, Leu423, Lys660, Ala680, Tyr729 and Arg781 labeled. (b) The ELAC2 model is rotated with linker on left with the residues Arg68, Gln388 and Arg564 labeled. (c) ELAC2 rotated with linker on right and the residues Pro493, Thr520 and His749 labeled. Note: the residues at Arg68, Phe154, and Gln388 in H. sapiens ELAC2 map to the domain interface

Figure 5 In silico analysis of disease‐related ELAC2 substitutions p.Pro493Leu and p.Tyr729Cys. (a) Structure overview of S. cerevisiae Trz1. Amino and carboxy domains and linker are colored as in previous figures. The curved arrow indicates the presumed substrate trough. Metal ions which mark the active site are shown as blue spheres. PxKxRN and Motif V loops are shown in magenta and orange, respectively. (b) Detailed view of PxKxRN loop (magenta) and Motif V loop (orange) in S. cerevisiae Trz1. The conserved basic residues Lys in the PxKxRN loop and Arg in the Motif V loop are shown as sticks, with H. sapiens equivalent residues given in brackets. Red arc illustrates the path of substrate acceptor stem and 3′ trailer through the presumed substrate trough. (c) Detailed view of PxKxRN loop (magenta) and Motif V loop (orange) in B. subtillis with pre‐tRNA substrate (Note: The B. subtillis structure displays similar folds and relative orientations, validating its use for modeling ELAC2 (RNase ZL) structure including the carboxy domain and the active site. The B. subtilis RNase Z structure 4GCW (Pellegrini et al., 2012) is the only available structure of an enzyme—pre‐tRNA substrate complex). The pre‐tRNA substrate acceptor stem is clamped by polar contacts with K in the PxKxRN loop (Lys495 in H. sapiens, Lys480 in S. cerevisiae, Lys15 in B. subtilis) and Arg in the Motif V loop (Arg728 in H. sapiens, Arg763 in S. cerevisiae, Arg273 in B. subtilis), illustrated with bold dashed lines. A polar substrate acceptor stem clamp consists of Lys15 contacts with 2′ and 3′ O's of ribose +1 and Arg273 contacts with two backbone phosphate O's on nt 72 and one on nt 73. Counterintuitively, the basic R‐groups that form the substrate clamp do not point toward each other; both are oriented toward the right, but two polar contacts extend to the right from Lys15 while three polar contacts by Arg273 extend to the left toward subst