Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan mitochondrial tRNAs

Abstract

Metazoan complexity and life-style depend on the bioenergetic potential of mitochondria. However, higher aerobic activity and genetic drift impose strong mutation pressure and risk of irreversible fitness decline in mitochondrial (mt)DNA-encoded genes. Bilaterian mitochondria-encoded tRNA genes, key players in mitochondrial activity, have accumulated mutations at significantly higher rates than their cytoplasmic counterparts, resulting in foreshortened and fragile structures. Here we show that fragility of mt tRNAs coincided with the evolution of bilaterian animals. We demonstrate that bilaterians compensated for this reduced structural complexity in mt tRNAs by sequence-independent induced-fit adaption to the cognate mitochondrial aminoacyl-tRNA synthetase (aaRS). Structural readout by nuclear-encoded aaRS partners relaxed the sequence constraints on mt tRNAs and facilitated accommodation of functionally disruptive mutational insults by cis-acting epistatic compensations. Our results thus suggest that mutational freedom in mt tRNA genes is an adaptation to increased mutation pressure that was associated with the evolution of animal complexity.

Fig. 1. Increased sequence divergence, degeneration of structural determinants and loss of sequence identity elements in bilaterian mt tRNAs.

a Average length (top) and sequence changes (middle) of mitochondrial (orange) and prokaryotic/cytoplasmic (ct) tRNAs (blue). The average length of ct tRNAs ranges from 72–74 nt to ≥80 nt for tRNAs with long variable arm. While non-bilaterian mt tRNAs retain canonical length distributions, average sequence length of bilaterian mt tRNAs drops significantly (Supplementary Fig. 1b) relative to cytoplasmic counterparts, with ~65 nt (mean) in protostomes (p < 0.001) and low branching deuterostomes, and ~69 nt in vertebrates (p<0.001). In terms of sequence divergence in tRNAs (middle), non-bilaterian mt tRNAs exhibit moderate but significant increases to ~44% sequence changes compared with ~40% (p < 0.001) in ct tRNAs, likely linked to increased A/U content (Supplementary Fig. 1a). Bilaterian mt tRNAs accumulate substitutions further to ~64% and ~58% in protostomes and deuterostomes, respectively, compared with ~40% in ct tRNAs (p < 0.001). Again, protostomes and low branching deuterostomes show stronger divergence than vertebrates, possibly also linked to stronger increases in A/U content. Mt tRNA^Ala and mt tRNA^Ser/UGA are highlighted in dark and light yellow, respectively; ct tRNA^Ala and ct tRNA^Ser/UGA are highlighted in dark and light blue, respectively. Each data point corresponds to one tRNA identity (Ala, Arg, etc.) within phylogenetic groups indicated below the x-axis. Sequence divergence was determined relative to α-proteobacterial tRNAs. The bottom panel shows the phylogenetic relationship and divergence times between the organismal groups for which tRNAs were compared. P-values were determined by Wilcoxon rank sum test (Supplementary Fig. 1b). Source data are provided as a Source Data file. b Scheme of cloverleaf and L-shaped fold of canonical tRNA with conserved structure- and sequence elements. Canonical tertiary interactions are indicated by cyan dashed lines. c Examples of mt tRNA^Ala (orange) from Octopus vulgaris (Ov) and humans (Hs) and of canonical tRNA^Ala (blue) from E. coli and human cytoplasm. Red boxes indicate the G3:U70 identity element. In O. vulgaris and human mt tRNA^Ala, G3:U70 is replaced by U:A and G:C pairs, respectively (bold red) (see also Supplementary Figs. 1–3).

Fig. 2. Sequence-independent recognition of Hs mt tRNA^Ala by mt AlaRS.

a Hs mt tRNA^Ala mutants used in charging experiments with mt AlaRS. b–d Relative charging activities (k_cat/K_m) of mt AlaRS on mt tRNA^Ala variants (activity with wild-type mt tRNA^Ala is set to 1). e Sequence of Hs ct tRNA^Ala (left) and relative activities of chimeric tRNA^Ala variants. Domains are colored orange for mt tRNA^Ala and blue for ct tRNA^Ala. Error bars represent SEM of two independent experiments for each wild-type and mutant. Source data are provided as a Source Data file (see also Table 1).

Fig. 3. Compensatory epistasis in Hs mt tRNA^Ala.

a Hs mt tRNA^Ala is presented in schematic L-shape. Shown is a subset of constructs used in this study that showed compensatory epistasis between sites. Red indicates initial mutations that reduce charging. The positions for compensatory mutations that restore charging are shown in green and are indicated by arrows. “T. obscurus” and “P. coquereli” denote the mt tRNA^Ala’s from two primate species that contain known human disease-causing mutations in an overall altered sequence context. Black circles indicate positions with sequence alterations relative to wild-type (WT) Hs mt tRNA^Ala with unknown effects on stability. b Relative charging levels for mt tRNA^Ala constructs containing the destabilizing mutation alone or in combination with compensatory mutations. The charging level of wild-type Hs mt tRNA^Ala is set to 100%. Error bars represent the SEM of two to six independent experiments. Source data are provided as a Source Data file (see also Supplementary Figs. 4–6 and Table 2 for data on the complete set of compensatory mutations used in this study).

Fig. 4. mt tRNA^Ala recognition by mt C-Ala.

a Structures of Hs mt C-Ala (left), Hs ct C-Ala (middle), and A. fulgidus C-Ala (right). b Structure model of mt tRNA^Ala (orange) bound to Hs mt AlaRS. The crystal structure of mt C-Ala is shown in cyan. Red and blue arrows indicate views in (c) and (d), respectively. c, d Details of the possible interaction interfaces between mt C-Ala and tRNA as indicated by the model. c Interaction of T-stem-loop (orange) and V-loop (cyan) with the N-terminal subdomain. d Interaction of T-loop and D-loop (orange) with the globular subdomain. In (c), the additional V-loop nucleotide that is absent in mt tRNA^Ala but present in ct tRNA^Ala (as U47), is shown in transparent gray. e Impact of mutations in Hs mt AlaRS on mt tRNA^Ala charging. Error bars represent SEM of triplicate experiments. Source data are provided as a Source Data file. f Comparison of surface charge distributions in Hs mt C-Ala (left) and Af C-Ala (right). Blue indicates positive, red is negative surface charge. Arrows indicate the position of the V-loop. g Superposition of Hs mt C-Ala (cyan) and Af C-Ala (yellow) based on the C-terminal globular domain (see also Supplementary Figs. 7 and 9; see Supplementary Table 1 for data collection and refinement statistics).

Fig. 5. Structural idiosyncrasies and instability in the tRNA elbow region act as anti-identity elements between mtaaRS/tRNA systems.

a Hs mt AlaRS charges cognate mt tRNA^Ala but not mt tRNAs specific for Asp, Leu, or Ser. Error bars represent the SEM from triplicate experiments. b Cloverleaf presentations of Hs mt tRNA^Asp (left) and variants containing D- and T-stem-loop and V-loop elements from mt tRNA^Ala (orange) or canonical ct tRNAs (blue/purple). Dashed lines indicate the potential for canonical D-loop-T-loop tertiary interactions. c Charging activity of Hs mt AlaRS on mt tRNA^Asp variants relative to activities on wild-type mt tRNA^Ala (blue; top y-axis) and wild-type mt tRNA^Asp (gray; bottom y-axis). See also Table 3. Error bars represent SEM of two to six independent experiments. Source data are provided as a Source Data file. d Hs mt SerRS charges cognate mt tRNA^Ser/UGA and stabilized mt tRNA^Asp variants but not wild-type mt tRNAs specific for Ala or Asp. Error bars represent the SEM from triplicate experiments. e Thermal melts for wild-type and stabilized mt tRNA variants and Hs ct tRNA^Ala (see also Supplementary Fig. 10).