Peer review of: Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

The manuscript deals with the aspect of how mitochondrial translation (tRNA and aaRS) system 
adapts to the high mutational rate of the mitochondrial genome. Further, the authors have performed 
bioinformatic and biochemical studies based on which they claim that a G3:U70-independent 
recognition of tRNAAla is facilitated by key adaptations at the mitochondrial AlaRS level. Interestingly, 
they show that the aminoacylation barrier can be overcome by transplanting the variable arm (in 
particular the elbow region) thus identifying a mitochondrion specific tRNA identity element for 
tRNAAla system. 
Overall, the manuscript is trying to link the above experimental observations to a larger phenomenon 
of mutational freedom in mt tRNAs which is possibly linked to the evolution of bilaterians. In my 
opinion, the claim is too tall and not sufficiently supported (see below). Also, the authors choose 
systems and examples arbitrarily to make their point while conveniently leaving out the exceptions! 

Major concerns: 
a) One of the major claims of the article is ‘bilaterians mt tRNA have lost the identity determinants 
and are unstable’. 
• The unusual size and structure of mt tRNAs were previously noted (Suzuki et al, Annual Review of 
Genetics 2011, 45:1, 299-329; Salinas-Giege et al, Int. J. Mol. Sci. 2015, 15, 4518-4559). 
• Is there any rationale to analyse only 9 out of 20 amino acid-specific tRNAs? Supplementary Figure 
1 includes tRNAs only from primates and mammals. What is the scenario with respect to lower 
bilaterians and non-bilaterians mt tRNAs? 
• The loss of identity determinants in mt tRNAAla for AlaRS charging was reported in previous studies 
and an alternate mode of recognition was proposed. The G3:U70-independent mode of recognition by 
mt AlaRS was beautifully shown very recently by En-Duo Wang and co-workers, wherein multiple 
positions on the tRNA are recognized by the mitochondrial AlaRS (Zeng et al, Nucleic Acid Research 
2019, 47:6, 3072-3085). 
• The authors claim that G3:U70 is entirely lost from the metazoans mitochondria. In contradiction to 
this, it has been previously (Chihade et al, Biochemistry 1998, 37:25, 9193-9202) shown that C. 
elegans mitochondrial AlaRS uses G3:U70 as a determinant for charging the mt-tRNAAla. Like C. 
elegans, other bilaterians such as Albinaria caerulea and Ascaris suum also have a G3:U70 in the mt-
tRNAAla. Hence the metazoan/bilaterian based argument does not hold good and unfortunately the 
authors neither choose to point these out or provide an explanation for these violations. 
• Previous work from the same group has shown that Drosophila melanogaster mtAlaRS recognizes 
the translocated G2:U71 of mt tRNAAla, while G3:U70 acts as a negative determinant for 
aminoacylation (Lovato et al, EMBO 2001, 20:4846-4853). Hence, the universality of G:U recognition 
in tRNAAla was put forward using the above data from the mitochondrial system as well. In light of 
these studies, the argument of bilaterian specific phenomenon becomes fragile and needs to be 
revisited. 

b) The second major claim is the ‘molecular adaptation of mtAlaRS to recognize the elbow region of 
the tRNA by creating a positive patch in the C-Ala domain, which is essential for aminoacylation’. 
• The mutational analysis of the C-Ala domain (positive patch) shows that K822, R853, R860 are 
important for recognizing the tRNA. However, these residues are expected to be specific to mtAlaRS 
only, but (from supplementary figure 5) these residues are also conserved in ctAlaRSs. Thus, the 
claim for the appearance of the positive patch only in C-Ala domain of bilaterian mtAlaRS is 
questionable! 
• R826 of mtAlaRS is not conserved but mutation to Alanine does affect the aminoacylation rate in one 
case. Does that mean that the proposed model is specific to only a very small subset of higher 
organisms? 
• The authors present the sequence alignment of AlaRS from different organisms. However, for 
mtAlaRS the list includes only chordates while leaving out the lower bilaterians/metazoans. 

• As the structure solved here is only the C-Ala domain and the interpretation is based on the full-
length model stating “In particular, the mt AlaRS-specific Gly825 allows tight association between C-
Ala and tRNA” may not be appropriate. Do the authors mean that introducing G825 (along with M343, 
G444) in ctAlaRS is sufficient enough to charge mt-tRNAAla? 

c) The authors generalize the mechanism of “chaperon-like fitting” to the rest of the mt synthetases. 
• A major premise of this manuscript is “We hypothesized that, in contrast to the rigid lock-key 
selection of cytoplasmic AARS-tRNA pairs, an adaptive chaperone-like fitting of the nuclear encoded 
synthetase with its mt tRNA partner made possible the mutational freedom of mt tRNAs”. Due to the 
multidomain architecture of aaRSs, they are shown to be involved in an induced-fit mode of enzyme-
substrate interaction (Sherlin & Perona, Structure 2003, 11: 591–603). The induced fit/conformational 
selection of tRNA-synthetase recognition is so well known in the field and therefore it is difficult to 
imagine that the authors choose to present a totally different narrative! 
• In abstract the authors state “Outsourcing structural stability to nuclear-encoded aaRS partners” 
which is incorrect. By using the term “Outsourcing” does the authors mean the stability of mt aaRS is 
more compared to ct aaRS? Since the complex possibly is more stable, there is no ‘outsourcing’ 
happening here! 
• As correctly quoted (line 208), earlier studies have shown that mtSerRS uses tRNA elbow region for 
its charging. As the entire experimental proof for the current hypothesis is based on AlaRS and SerRS 
which do not follow the usual anticodon based tRNA recognition, any extrapolation or generalization to 
the rest of the mt synthetases may not be appropriate. 
• Earlier studies have shown that in case of mitochondria the number of determinants for 
aminoacylation are reduced but not rewired fully (Suzuki et al, Annual Review of Genetics 2011, 45:1, 
299-329). 

d) tRNA sequence-independent recognition of mt tRNAAla by mt AlaRS. 
• In line 137, the authors contradict the recent finding (Zeng et al, Nucleic Acid Research 2019, 47:6) 
and state that the tRNA recognition by mtAlaRS is based on the structure but not the sequence of the 
mt tRNAAla. Given the inadequate structural and biochemical data available, it is premature to say 
that tRNA structure plays an important role but not sequence. Perhaps, it appears that it is the 
combination of both which is recognized by mtAlaRS. In any case, the current work does not go 
beyond what has been published by Zeng et al. NAR 2019 in this aspect. 

Other concerns: 
• Line 23: ‘aminoacyl tRNA’ should be written as ‘aminoacyl-tRNA’ in the abstract. 
• Line 26: ‘Or’ needs to be replaced by ‘Our’ in the abstract. 
• Line 136: The non-reverting mutation of mt tRNAAla U54/58 is not shown in Figure 3c or table1-3. 
• In Figure 5c the activity is plotted relative to tRNAAsp, instead, these values need to be compared 
with mt tRNAAla. 
• Line 210: The authors do not include Ser in the earlier bioinformatics analysis (Line 70), but use 
them as a proof of concept. 
• Here the authors use SerRS as a proof of concept. Is it possible to show with other more canonical 
mt synthetases? 
• Line 502: The reference volume and page numbers are incorrect. 
• Table 2: the tRNA mutation ‘G1:C73/C62/C68’ is incorrect, and should be changed to 
‘G1:C72/C62/C68’ 

Reviewer #2 (Remarks to the Author): 

This important manuscript describes new phenomena relevant to understanding how the emergence of 
mitochondria as an efficient energy source impacted the evolutionary emergence of higher-order 
animals with bilateral symmetry. The notional steps in the argument are these: (i) mitochondria 

reproduce asexually and without recombination, making them potentially vulnerable to irreversible 
accumulation of deleterious mutations (i.e. Muller’s ratchet); (ii) mitochondrial transfer RNAs exhibit 
extraordinary variations in size and sequence, compared to the more stable cytoplasmic tRNAs; (iii) 
mitochondrial tRNA variants are both structurally less complex and relatively unstable; (iv) the 
cognate mitochondrial aminoacyl-tRNA synthetase structures—encoded in the nucleus—compensate 
these vulnerabilities by developing unusual induced-fit binding mechanisms quite distinct from those 
of the cytoplasmic cognate pairs. As each of these statements opens substantial questions, the 
manuscript is necessarily highly speculative. Nonetheless, the authors do a credible job shoring up 
each conclusion. 

The manuscript combines several diverse threads from the Schimmel laboratory, including their 
interest in disease-causing mutations mitochondrial tRNAs, significant markers of the major branch 
points in the evolution of higher organisms, and their long-standing contributions to the structural 
enzymology of aminoacyl-tRNA synthetases. The paper should be highly interesting to a broad 
spectrum of readers. 

The work described was motivated by detailed studies of the disease-causing mutations in the 
mitochondrial tRNAAla. The salient conclusion from an impressive number of observations and related 
experiments in this manuscript is that these disease-causing mutations can be compensated by 
mutations at other sites (epistasis) as well as by transformed binding interfaces with the cognate 
synthetase. In particular, chimeric tRNAAla molecules in which regions of the cytoplasmic tRNAAla are 
replaced by their counterparts from mitochondrial tRNAAla restored the ability to acylate the 
cytoplasmic chimeras. Because these experiments suggested that context, rather than sequence 
determined binding, an induced-fit binding mechanism involving the elbow region appears critical for 
productive binding. 

The authors describe structural data pertinent to this phenomenon by solving the domain responsible 
for binding the elbow from the mitochondrial synthetase, and combine it with artful homology 
modeling to show the extensive co-adaptation that shifted the recognition mechanism from a highly 
focused and sequence-dependent recognition of the (G3:U70) base pair, used almost universally by 
AlaRSs, to a more sequence-independent recognition of the tRNA elbow in the human mitochondrial 
AlaRS•tRNAAla complex. 

Similar, but more limited analysis of mitochondrial tRNASer reveals comparable changes, suggesting 
that the mitochondrial alanine system is representative of a detailed distinction between how tRNAs 
are recognized by cytoplasmic aminoacyl-tRNA synthetases and (nuclear encoded) mitochondrial 
synthetases. An imaginative speculation by the authors is that the nuclear synthetase genes for 
mitochondrial synthetases co-adapt to the rapid sequence evolution of mitochondrial tRNA synthetase 
genes by developing chaperone-like induced-fit recognition mechanisms that preserve the coding 
specificity necessary to ensure survival of the bilateran energy-generating mitochondria, whose 
genomic DNA must evolve under higher oxygen tension. 

The paper is quite well-written and conclusions are fully documented. The authors might consider the 
following suggestions: 

1. P. 2, line 26. “Or” should probably be “Our”. 
2. P. 2, line 27. “evolution of animal complexity”. Are there important differences between the 
bilateran mt tRNA sequences and those from the plastids of other higher organisms (i.e. chloroplasts). 
3. P. 3, lines 59-62. The authors run the risk of making to sharp a distinction here between “lock and 
key” and induced-fit. Most of the cytoplasmic synthetases are likely to exhibit induced fit in various 
ways as well, so referring to them categorically as “lock and key” may eventually backfire. 
4. P. 5, lines 105-109. Not clear what the distinction is between "reduced charging" and "mainly due 
to decreased overall yields of charged tRNA.” 
5. P. 9, lines 191-192. The dramatic destabilization of mitochondrial tRNAs might usefully appear 

much earlier in the development, in particular in the introduction. The same holds for the fact that the 
mitochondrial alanyl-tRNA synthetase doesn’t use the (G3:U70) (Page 8, line 175) base pair as an 
identity element. I found it troubling to be told these two, quite salient fact, so late in the paper. 
6. P. 11, line 226. Is “for” supposed to be “from”? 
7. P. 12, lines 250-254. These two sentences try to say too much, and I cannot exactly follow what 
the authors really want to say, despite the fact that it is closely related to their overall message. It is 
difficult to unpack the conflation of induced fit and negative determinants. 

Charlie Carter, Professor of Biochemistry and Biophysics, UNC Chapel Hill 

Reviewer #3 (Remarks to the Author): 

Summary: 

In the manuscript "Relaxed sequence constraints favor mutational freedom in idiosyncratic metazoan 
mitochondrial tRNAs", the authors formulate a model by which the extreme divergence of 
mitochondrial tRNAs in both sequence and structure, relative to their cytosolic counterparts. In their 
model, the deleterious effects of the inevitable accumulation of substitutions in mitochondrial tRNAs 
via Muller's ratchet are offset by the evolution of the nuclear-encoded mitochondrial synthetase to be 
more conducive to a broad array of mitochondrial tRNA sequences. The synthetases here maintain 
their specificity through recognition of structural elements of mitochondrial tRNA transcripts, rather 
than focusing on tRNA sequence features that cannot be expected to be conserved due to the lack of 
recombination of mitochondrial genomes. Overall, the authors do well to identify a system consistent 
with the divergence of mitochondrial tRNA genes, which is striking in comparison to cytosolic tRNA 
genes. Their model, as well as functional analyses to identify epistatic compensatory substitutions and 
their effects on tRNA charging, are well executed. 

This manuscript introduces a unique and important example of cooperation between mitochondrial and 
nuclear gene sequences, and highlights the differences in modes of evolution of the two. It is a good 
fit for the journal and of general interest to readers. 

Primary Concerns: 

However, there are some concerns about potentially confounding factors that are not addressed: 

1) The mitochondrial import of cytosolic tRNA transcripts is widespread across taxa (e.g. reviewed in 
Salinas-Giegé, et al, Int. J. Mol. Sci. 2015). This is particularly important if bilaterians import tRNAs at 
higher rates than their ancestors. This is consistent with some aspects of the model, including 
potentially decreased selective constraints on mitochondrial tRNAs to maintain their sequence 
integrity. However, this might also place decreased constraint on the mt-tRNA and the mt-AARS to be 
in sync with each other. Mitochondrial import should therefore be addressed in the manuscript. 

2) Several mitochondrial tRNA genes do not exhibit the bizarre structural divergence highlighted here 
and instead are quite similar to cytosolic tRNA genes. There are some hypothetical mechanisms likely 
consistent with the authors’ model that could be mentioned (differential mutation rates by locus, less 
stringent rules for recognition by certain AARSs). The focus throughout on tRNA-Ala does well as a 
clear example of the observed divergence, but addressing that many mt-tRNAs are exceptions to the 
model, or alternatively, predicted by the model due to other factors, would make this more impactful. 

3) The manuscript points at an increased level of sequence divergence beginning at the Bilaterian 
divergence, but the figures make this look more like a peak at this point before trending back 
downwards. This pattern should be addressed. More generally, Figure 1 is pretty challenging to parse. 

1b seems to show sequence divergence from the tips of the tree at the node connecting to other 
lineages. At a glance, one would assume these are reconstructed ancestral states, but they are not. 
Additionally, it is not clear how the estimates of nucleotide divergence and length changes map onto 
the tree at the bottom. I assume some lineages are not included, but it is not entirely clear. I 
recommend a significantly reworked figure and a much more detailed legend. One way this can be 
done is to have each lineage on the tree terminate at the present and then to display all relevant data 
for each lineage to the right of the branches. 

4) The word "significantly" is used several times in your manuscript without reference to a statistical 
test or p-value. Most importantly, the point addressed by Figure 1 would be benefitted by e.g. a 
Wilcoxon Rank Sum test to demonstrate that mt-tRNAs show greater divergence following bilaterian 
expansion, or a Spearman Rank correlation to correlate divergence time and mt-tRNA sequence 
divergence, depending on the intended message. This is important to demonstrate because it is not 
completely clear based on the relatively greater abundance of data points after bilaterians that the 
stated trend is significant or that it continues through time. 

5) The section "Mutational freedom associates with low complexity sequences" would benefit from 
reduction. It would be best if this section focused on a few of the most exemplary or interesting 
epistatic effects, and the rest could be moved to a supplemental section. The general readership of 
Nature Communications is unlikely to find each example sufficiently compelling to merit this extended 
treatment. 

Minor Concerns: 

1) In Figure 1, it would be best to assign a letter to each panel so as not to have to refer to portions 
as e.g. "the inset in the middle panel of Figure 1d". An explanation should also be provided on 
whether there is any significance for the branch lengths of e.g. Archaea, Bacteria, or whether only the 
divergence time (and not branch lengths) is meant to be indicated by this figure. 

2) Figure 2 would benefit from being simplified. I would recommend only showing the WT, each of the 
six deletions, and the epistatic substitution(s) that restored each mutation genotype back to WT 
charging level. The rest would be better suited as a supplemental figure. 

3) The paper concludes (lines 264-267) with a fairly specific order of events. The idea presented in the 
final paragraph of the manuscript is that mutational burden increased, and deleterious mutations in 
tRNAs selected for the evolution of aaRS recognition. However, it is also possible that the aaRS 
recognition evolved first and permitted the degradation of tRNA sequence via nearly-neutral 
divergence. The authors should consider being more circumspect in their explanation. 

4) It would be beneficial to include some aspects of mitochondrial inheritance that differ from what is 
assumed under most cases of Muller's ratchet. For example, many cells contain multiple mitochondria, 
and there are quality control mechanisms including purifying selection to reduce the chances of 
transmitting harmful mtDNA to offspring (e.g. reviewed in Mishra and Chan, Nature Reviews, 2014). 
Differences in mitochondrial quality control across species may impact your model, and should be 
discussed.