A small protein coded within the mitochondrial canonical gene nd4 regulates mitochondrial bioenergetics

Abstract

Background Mitochondria have a central role in cellular functions, aging, and in certain diseases. They possess their own genome, a vestige of their bacterial ancestor. Over the course of evolution, most of the genes of the ancestor have been lost or transferred to the nucleus. In humans, the mtDNA is a very small circular molecule with a functional repertoire limited to only 37 genes. Its extremely compact nature with genes arranged one after the other and separated by short non-coding regions suggests that there is little room for evolutionary novelties. This is radically different from bacterial genomes, which are also circular but much larger, and in which we can find genes inside other genes. These sequences, different from the reference coding sequences, are called alternatives open reading frames or altORFs, and they are involved in key biological functions. However, whether altORFs exist in mitochondrial protein-coding genes or elsewhere in the human mitogenome has not been fully addressed. Results We found a downstream alternative ATG initiation codon in the + 3 reading frame of the human mitochondrial nd4 gene. This newly characterized altORF encodes a 99-amino-acid-long polypeptide, MTALTND4, which is conserved in primates. Our custom antibody, but not the pre-immune serum, was able to immunoprecipitate MTALTND4 from HeLa cell lysates, confirming the existence of an endogenous MTALTND4 peptide. The protein is localized in mitochondria and cytoplasm and is also found in the plasma, and it impacts cell and mitochondrial physiology. Conclusions Many human mitochondrial translated ORFs might have so far gone unnoticed. By ignoring mtaltORFs, we have underestimated the coding potential of the mitogenome. Alternative mitochondrial peptides such as MTALTND4 may offer a new framework for the investigation of mitochondrial functions and diseases.

Mitochondrial polymorphism m.3017C>T of SHLP6 relates to heterothermy

“…Recently, the use of translation from alternative translation initiation codons applied by the mitochondrion got a renaissance, for example, illustrated by the mitochondrially encoded human peptides MTALTND4 and SHLP6 ( Kienzle et al, 2023 ). In the latter case, the authors assumed an involvement of the alternative starts codons ATT and ATA to expand the peptide (33 instead of the canonical twenty amino acids).…”

“… Sporadic detection of the non-enriched human SHLP6 using MS analysis (A) Hypothetical extension of the canonical human 20mer of SHLP6 predicted by Kienzle et al (2023) . This putative N-terminal extension is depicted by smaller font type.…”

“…The common mitogenome in Vertebrata encodes 13 essential protein subunits of the respiratory chain, two rRNAs, 22 tRNAs ( Ren et al, 2023 ) and several small open-reading frame (sORF)-derived micropeptides, commonly defined as polypeptides with a relatively arbitrary length of less than 100 to 150 amino acids [references in ( Sousa and Farkas, 2018 )], that represent the low end of the canonical protein spectrum ( Schlesinger and Elsasser, 2022 ). Currently, the latter subgroup of micropeptides termed MDPs, consists of Humanin ( Hashimoto et al, 2001 ), Gau [gene antisense ubiquitous ( Faure et al, 2011 )], MOTS-c [mitochondrial ORF of the 12S rRNA type-c ( Lee et al, 2015 )], SHLP1-6 [small Humanin-like peptides 1 to 6 ( Cobb et al, 2016 )], SHMOOSE [Small Human Mitochondrial ORF Over SErine tRNA ( Miller et al, 2022 )] and MTALTND4 [mitochondrial alternative ND4 protein ( Kienzle et al, 2023 )]. At least for a species which does not have a nuclear insertion of mitochondrial origin [NUMT ( Wei et al, 2022 )], mtDNA is the exclusive source for MDP coding, as, for example, in the cases of rat Humanin and rat MOTS-c ( Paharkova et al, 2015 ).…”

“…At least for a species which does not have a nuclear insertion of mitochondrial origin [NUMT ( Wei et al, 2022 )], mtDNA is the exclusive source for MDP coding, as, for example, in the cases of rat Humanin and rat MOTS-c ( Paharkova et al, 2015 ). Cytoplasmic tRNA import ( Rubio et al, 2008 ; Jeandard et al, 2019 ; Kienzle et al, 2023 ) and the use of alternative start codons (compiled by NCBI at https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi ) expand the coding capacity of the mitochondrial genetic code, with consequences for MDP size (see below, Figures 3 , 4F ) and also for fine-tuning of complex biological systems that would be highly likely given the uniquely suited small size of MDPs.…”

Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk

“…The human mitoribosome translates at least 13 respiratory chain protein-coding mRNAs from the mitochondrial genome 1 . It consists of three rRNAs and at least 82 mitoribosomal proteins, 36 of which have no corresponding homologs in bacterial and cytosolic ribosomes.…”