Multiple origins of a frameshift insertion in a mitochondrial gene in birds and turtles

Abstract Background During evolutionary history, molecular mechanisms have emerged to cope with deleterious mutations. Frameshift insertions in protein-coding sequences are extremely rare because they disrupt the reading frame. There are a few known examples of their correction through translational frameshifting, a process that enables ribosomes to skip nucleotides during translation to regain proper reading frame. Corrective frameshifting has been proposed to act on the single base pair insertion at position 174 of the mitochondrial NADH dehydrogenase subunit 3 gene (ND3) that has been observed in several turtles and birds. However, the relatively sparse taxonomic representation has hampered our understanding of the evolution of this insertion in vertebrates. Results Here, we analyzed 87,707 ND3 sequences from 10,309 vertebrate taxa to reveal the evolutionary history of this insertion and its common genomic characteristics. We confirmed that the insertion only appears in turtles and birds and reconstructed that it evolved independently in both groups with complex patterns of gains and losses. The insertion was observed in almost all bird orders but was absent in all members of the diverse Passeriformes. We found strong conservation in the nucleotides surrounding the insertion in both turtles and birds, which implies that the insertion enforces structural constraints that could be involved in its correction. Conclusions Our study demonstrates that frameshifts can be widespread and can be retained for millions of years if they are embedded in a conserved sequence theme.

When checking for this taxon, I noticed that (i) it is present twice in the alignment, likely due to the comma present in one of the labels, that likely made it escape the filtering to keep single sequences per taxon; perhaps this is also true for other taxa, thus adding redundant information ; (ii) this insertion is absent in the alignment and its incorrect filtering (maybe it is present in less than 95% of the sequences?) results in disrupted reading frames for the rest of the species. In order to verify, I checked the mitogenome in Genbank (NC_001947.1), where the insertion is explicitly annotated as an exception and present in the annotated sequence (see captions of nucleotide and protein alignments in joint pdf file). I consider that including it, along with the possibility that it happens elsewhere in the phylogeny, is susceptible to change the big picture of the evolution of insertions in this gene and should be addressed in this manuscript. I think it would also be interesting some analyses on tRNAs, as coping with the frame-shifts described in this manuscript could be reflected in tRNA structures (see, for example, Haen et al (2014)). This option is not mentioned in the manuscript and given that some species present more than one insertion in coding genes along the mitogenome, it should definitively be considered. Given that a significant portion of the sequences in this study comes from fully assembled and annotated mitogenomes (e.g. RefSeq sequences from Genbank), having a look for differences in tRNAs sequences and structures should be feasible and would provide more value and support to the inferred trends and observations. References on mechanisms to cope with frameshifts Seligmann, H., Warthi, G.

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