Peer review of: Complete chemical structures of human mitochondrial tRNAs (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

tRNAs are central to the decoding process and the post-transcriptional modifications of tRNA are critical 
for tRNA function including stability, folding and decoding. It is known that disorders of mitochondrial 
translation may lead to the mitochondria dysfunction and some point mutations in mt-tRNA have been 
reported to be associated with mitochondrial diseases. But the molecular pathogenesis of human 
mitochondrial diseases caused by aberrant tRNA modifications has lagged behind due to the lack of the 
complete landscape of modifications in mt-tRNA and the list of responsible modifying enzymes. In this 
manuscript, Suzuki and his colleagues identified the majority of modifications in all 22 human mt-tRNA 
using mass spectrometric analyses. Further, a comprehensive analysis including CE-Ψ-MS, CMC-PE and 
tRNA-Ψ-seq were employed to detect Ψ (a mass-silent modification) in mt-tRNA. In addition, the 
modifying enzymes responsible for these mt-tRNA are also listed. Thus, the collective results represent a 
very comprehensive resource that will be a key reference for the field. The study will certainly benefit 
future molecular mechanism studies of human mitochondrial diseases caused by mt-tRNA 
modifications. The study is also very well designed, performed and written. Hence, I strongly 
recommend the publication of this paper at Nature communications. I only have a few minor points as 
listed. 

1. In this study, tRNA-Ψ-seq was used to detect Ψ sites in mt-tRNA. Recently a similar approach named 
DM–Ψ-seq is also developed and identified Ψ sites in cytoplasmic and mitochondrial tRNA (Song et al., 
2020, Nat Chem Biol 16, 160-169). It would be nice to also compare the psiU sites (since it is a mass-
silent modification) identified in this study with the reference. 

2. The authors have listed RNase T1- or A-digested fragments of 22 human mt-tRNAs in supplementary 
table 1; all the original fragment analysis maps (as Figure 2C) are also desired to be included, which can 
be used to evaluate the stoichiometry of modified nucleosides. 

3. 2D-TLC cannot discriminate Ψ in different positions of mt-tRNAGlu and hence the label in 
Supplementary 4 is not accurate. 

Reviewer #2 (Remarks to the Author): 

In this study, Suzuki et al, performed a comprehensive analysis of post-transcriptional modifications of 
all human mt-tRNAs. In a previous analysis, the authors mapped 15 types of modified bases at 118 
positions in all species of bovine mt-tRNAs. Here, the authors isolated 22 species of human mt-tRNAs 
and analyzed the primary sequence and post-transcriptional modifications for each tRNA using mass 

spectrometry in combination with biochemical analyses to map pseudouridines. In total, the authors 
found 16 kinds of RNA modifications at 136 positions in 22 species of human mt-tRNAs. The results 
provide a complete list of modifications in human mt-tRNA. This is an interesting study and should 
facilitate investigations of their physiological roles in the future. 

1. The authors isolated 22 species of human mt-tRNAs from human placenta, but only several species of 
mt-tRNA from HeLa cells. Why not isolate the all 22 species of human mt-tRNA from HeLa cells? I 
suggest the authors to compare the modifications in mt-tRNA between human placenta and HeLa cells. 

2. How about the frequency (or stoichiometry) of specific modifications in given sites of human mt-
tRNA? I suggest the authors to provide this information for each identified modification in mt-tRNA. 

3. How to confirm the purity of the isolated individual mt-tRNA? The lengths of mt-tRNA are similar and 
they also show similar gel-shift on the gel. Single band in gel may not indicate the single mt-tRNA. The 
impure mt-tRNA may affect the determination of the modifications in mt-tRNA. 

4. There are some modifications that have the same molecular weights, such as m5C and m3C, m1G and 
m2G, m1A and m6A. How to confirm these modifications? The authors need to provide detailed 
experimental evidence. 

5. The MS fragment ions of 15 species of mt-tRNA were listed in Table S1. But in this study the authors 
measured 22 species of mt-tRNA. The authors need to supply the rest part of mt-tRNA of the MS 
fragment ions. 

6. The authors need to provide the sequence and Western blotting data for supporting the knockout 
experiments. 

7. In Figure 3b, the Ψs at positions 66 and 67 in human mt-tRNAPro were identified. If these two sites 
are both Ψs, why there are two possibilities, i.e. the existence of c6 and y6 ions? 

8. Figure 4 and Figure S5 are somehow similar. Figure 4 includes 14 species of mt-tRNA and Figure S5 
includes 22. The authors may only provide the full 22 species of mt-tRNA in main text. 

Reviewer #3 (Remarks to the Author): 

In this manuscript by Suzuki et al., the authors map the chemical structures of the remaining 
uncharacterized tRNAs encoded by the human mitochondrial genome. Using previously-developed tRNA 
purification and mass spectrometry techniques, the authors identify the repertoire of modifications in 
human mitochondrial tRNAs. The data is technically sound and the authors provide independent assays 
to validate a subset of modifications. Moreover, the authors provide evidence that the QTRT1 and 
QTRT2 proteins are required for the queuosine modification in certain human mitochondrial tRNAs. The 

manuscript is clearly written and have provided extensive Supplemental Data to support their findings. 

Unfortunately, there is a lack of novelty in this manuscript since no new types of modifications were 
found. The approach and results are similar to the conclusions published by the group using bovine 
mitochondrial tRNAs in their 2014 paper in Nucleic Acids Research. Thus, many of the modifications in 
mitochondrial tRNA could have been predicted and this manuscript serves as validation rather than 
discovery. Moreover, there is little functional characterization of the modifications and their biological 
roles. Table 1 is just a list of codons and the cognate tRNA anticodons while Table 2 is a list of the known 
and confirmed tRNA modifications and their cognate enzymes. While the results represent a complete 
inventory of human mitochondrial tRNA modifications, no new insight is gained that changes how we 
think about mitochondrial tRNA modifications or mitochondrial tRNA biology. 

To elevate this manuscript beyond just simply mapping tRNA modifications, the authors should 
characterize the consequences of lacking a mitochondrial tRNA modification. For example, the Authors 
have made a knockout of QTRT1 or QTRT2 but there is no follow-up on the function of queuosine 
modification in mitochondrial tRNAs. Biological insight could also be gained by monitoring the function 
of the numerous pseudouridine modifications in mitochondrial tRNAs. Currently, the manuscript 
describes a list of modifications without providing any biological relevance or significance of the 
modifications. 

Minor points: 

• Figure 1 shows a denaturing gel of isolated tRNAs. Since the RNAs are imaged using a general nucleic 
acid stain, there is no follow-up on the relative purity of the isolated tRNA fractions. The Authors could 
perform Northern blotting on an aliquot of the isolated tRNAs to determine whether there are 
contaminating tRNAs in the fractions. This could be important since any contaminating tRNAs could 
contribute significantly to the modification signal detected by mass spectrometry. 
• As mentioned above, there is no follow-up on the QTRT1 or QTRT2-knockout (KO) cell lines. The 
addition of molecular or biological effects caused by lack of Q modification would have greatly 
strengthened the manuscript and provided insight into the function of the Q modification in 
mitochondrial tRNAs.