The functional importance of mitochondrial sequences with dual functions: overlapping protein–protein and protein–RNA gene sequences as test cases

Abstract Human mitochondrial DNA (mtDNA) contains 13 protein-coding subunits of the oxidative phosphorylation pathway, 22 tRNA and two rRNA genes. However, accumulating evidence suggests that mtDNA encodes additional overlapping genetic elements, including mitochondrial-derived peptides (MDPs) and alternative reading frames. Here, we assessed signatures of selection across 66 328 human mtDNAs and studied the potential impact of disease-causing mutation within these mtDNA overlapping sequences. By employing frame-specific dN/dS analysis for the overlapping reading frames, and codon position-specific diversity calculations we found that SHLP6 and SHLP3 (within 16S rRNA) display significant signatures of purifying selection. Mutational asymmetry analysis revealed purifying selection in both frames and strands of GAU/COX1, while other alternative reading frames show asymmetric patterns, supporting negative selection primarily on the encompassing canonical gene. Analysis of mito-ribosome profiling (HEK293 cells) revealed translation initiation signatures only for SHLP6 and ALTND4, providing functional support for their translation in HEK293 cells. Analysis of disease-causing mutations revealed that several such mutations have predicted deleterious effects on both canonical and alternative sequences, though canonical genes tend to be more frequently affected. Taken together, we provide evolutionary and functional evidence supporting biological relevance of certain MDPs and underline the need to re-evaluate the functionality of mutations in such sequences. This article is part of the theme issue ‘Evolutionary genetics of mitochondria: on diverse and common evolutionary constraints across eukarya’.