Ms by Rojas et al on Caretta mitochondrial tRNAs review by Herve Seligmann The ms presents bioinformatic analyses of structural variation in three mitochondrial tRNAs from Caretta caretta, a marine turtle under risk of extinction. The authors suggest that tRNA mutations are pathogenic. The ms is valuable but should be inproved before publication, along the lines developed below, and this in addition to improving the language, also beyond my own small comments.

My major comments fall into 4 main points, which would require little additional analyses, but would greatly expand the value of the ms. They relate to 3 functional properties of tRNAs.

1. Predicted aminoacylation of tRNA I suggest to analyze the tRNA sequences using TFAM (Taquist et al 2007, http://davidardell.org/software/tfam/). See if tendencies for correct/incorrect aminoacylations change for the various tRNAs. Along these lines, and using TFAM's output, I suggest to study the potential compensation between misacylation and codon-anticodon mismatches (Seligmann H BMC Mol Biol. 2010 May 28;11:41). The methods for this are described by Seligmann (Comput Biol Chem. 2011 Apr;35(2):81-95; Curr Genomics. 2012 Mar;13(1):37-54; Comput Biol Chem. 2016 Jun;62:119-32). Indeed, Seligmann (2011) shows that pathogenic mutations in Homo associate with weaker error compensation. Testing this for Caretta tRNAs could strengthen the ms's claims, and certainly the scientific impact of the study.

2. The complementary strand of tRNA genes might also template for functional tRNAs (Seligmann Biosystems. 2010 Jul;101(1):42-50; Biol Direct. 2010 Jun 16;5:39), and could have pathogenic implications (Seligmann J Theor Biol. 2011 Jan 21;269(1):287-96). This point should be at least discussed, could easily be explored via 2D structure predictions, and perhaps also 3D predictions. This is particularly relevant considering Seligmann's observations on marine turtle mitochondrial tRNAs (Comput Biol Chem. 2012 Dec;41:18-34). See also in this context Seligmann and Labra Biosystems. 2013 Dec;114(3):155-63.

3. The heavy strand sequences of mitochondrial tRNA genes might assist/replace the mitochondrial light strand repliction origin, OL (see various publications by Seligmann et al: J Theor Biol. 2006 Jul 21;241(2):321-32; J Exp Zool B Mol Dev Evol. 2006 Sep 15;306(5):433-49; J Theor Biol. 2006 Dec 7;243(3):375-85; J Mol Biol. 2008 May 23;379(1):188-99; Biosystems. 2010 Feb;99(2):85-93; Gene. 2014 Jun 1;542(2):248-57). It is easy to test (along methods described in these publications), whther the heavy strand tRNA sequences tend to form more or less OL-like seondary structures, and discuss the issue. Forming more or less OL-like structures affects the replicational mutations gradients, and is a probable factor for pathologies/effects on lifespan (Seligmann 2011 Mutation Patterns Due to Converging Mitochondrial Replication and Transcription Increase Lifespan, and Cause Growth Rate-Longevity Tradeoffs. Chapter 6 in "DNA Replication-Current Advances", book edited by Herve Seligmann, ISBN 978-953-307-593-8). See also Seligmann Curr Genomics. 2012 Mar;13(1):37-54.

4. tRNAs are not the only mitochondrial sequences with probable alternative functions, this is also the case for protein coding genes, rRNAs and Dloop (see for example: Faure E, Delaye L, Tribolo S, Levasseur A, Seligmann H, Barthélémy RM. Biol Direct. 2011 Oct 24;6:56. doi: 10.1186/1745-6150-6-56. Capt C, Passamonti M, Breton S. Mitochondrial DNA A DNA Mapp Seq Anal. 2016 Sep;27(5):3098-101; Breton S, Milani L, Ghiselli F, Guerra D, Stewart DT, Passamonti M.Trends Genet. 2014 Dec;30(12):555-64) and further publications by Seligmann (among others: Biosystems. 2012 Nov;110(2):84-106; J Theor Biol. 2015 Dec 21;387:154-65; Mitochondrial DNA A DNA Mapp Seq Anal. 2016 Jul;27(4):2440-6; Biosystems. 2016 Feb;140:38-48). It is important to remind that the known functions are not necessarily all functions.

The approach of this ms is commendable and should be followed by others.

Small comments. subdivide introduction and discussion into several paragraphs/sections line 46: when->went line 92: which species? this makes the section uncomprehensible. You do not list the species before making that statement. line 96: "is agree"-> "is in agreement" line 103: "exposed"-> "was uniform" or another expression, such as "presented no intraspecific polymorphism" line 111 "in"->"at" line 114 "motive"->motif? also in rest of text line 116 "one" delete lines 124, 181, 256, 258: complimentary->complementary lines 125, 256 "par"->pair line 139: basis?-> bases? nucleotides? line 145: "in"->at line 150: "the same as"->identical to lines 154, 156, 159: in->at line 158: "each one formed" reformulate sentence line 168: change sentence-> presented tertiary structure that differs from.... line 183:"basis pairs"->basepairs line 184: cause to distort-> cause distortions that perturb normal function (or something equivalent) line 185: bases pairs->basepairs line 186: reformulate sentence. "Most pathogenic mutations are in stems" line 201: "the modified" delete "the" lines 221,222, 240,283: "in"->at line 247: pars->pairs line 258: caused->formed line 261: microchondrial->mitochondrial line 262 delete "its" line 266: "mechanisms of repairing" -> repair mechanisms lines 267,269,273,276,289: trnaS line 283: "a" delete lines 329,397: Caretta caretta->italics line 369: "were"->sentence makes no sense

line 379: why conclusions after materials and methods, the latter after results? line 380: follows->delete s line 381: satisfies->satisfy Line 388: restructure last sentence