# Characterization of the Avian Mitochondrial-Derived Peptide MOTS-c and Its Potential Role as a Metabolic Regulator

## TL;DR

This study characterizes MOTS-c, a mitochondrial peptide in birds, revealing its unique sequence and potential role in regulating metabolism and gene expression.

## Contribution

The first comprehensive characterization of avian MOTS-c, including sequence divergence and functional insights into its metabolic regulatory role.

## Key findings

- Avian MOTS-c has a nucleotide deletion leading to divergent downstream amino acids compared to mammals.
- Chicken MOTS-c is highly expressed in the heart and reduced by fasting, suggesting a metabolic role.
- MOTS-c modulates the AKT signaling pathway in hepatocytes, linking it to metabolic regulation.

## Abstract

Mitochondria, key energy-producing organelles, critically depend on mitochondrial-encoded genes. This study systematically compared avian mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) coding/protein sequences and performed phylogenetic analysis. Avian MOTS-c sequences exhibit high similarity but contain a key nucleotide deletion compared with mammals, resulting in divergent downstream amino acids. Chicken MOTS-c is ubiquitously expressed, with the highest levels in the heart, and is significantly reduced by fasting. Integrating RNA-seq and molecular assays revealed that the chicken MOTS-c peptide modulates the AKT signaling pathway. These findings establish a foundation for exploring mitochondrial-derived peptides in birds and for informing mitochondrial genetic poultry breeding.

MOTS-c is a mitochondrial peptide that plays a crucial role in regulating energy metabolism, gene expression, and immune processes. However, current research primarily focuses on mammals like humans and mice, with no reports on avian MOTS-c. This study aimed to identify and characterize MOTS-c coding sequences across major poultry species through bioinformatics analysis and experimental validation. The alignment results showed high sequence similarity in the MOTS-c coding regions between avian and mammalian species. However, a single nucleotide deletion was identified in avian sequences at the position corresponding to the fourth amino acid residue of mammalian homologs, resulting in divergent downstream amino acid sequences. Despite this deletion, several residues were conserved across species. Phylogenetic analysis of mRNA sequences grouped pigeons with mammals, while protein sequence analysis revealed that poultry and mammals form separate branches, highlighting the divergence between avian and mammalian MOTS-c sequences. Tissue expression profiling demonstrated widespread distribution of chicken MOTS-c across multiple tissues, with the highest expression levels in the heart. Fasting significantly reduced heart MOTS-c expression, suggesting potential metabolic regulatory functions. Functional analysis of MOTS-c in primary hepatocytes revealed significant enrichment of the ribosome, oxidative phosphorylation, and key signaling pathways (PI3K-AKT and JAK-STAT) following 24 hours of treatment. Western blot validation confirmed MOTS-c-mediated activation of the AKT signaling pathway. This study represents the first comprehensive characterization of avian MOTS-c, providing critical insights into its evolutionary conservation and its potential functional roles in gene expression and cellular metabolism. Our findings establish a foundation for further investigation into the functions of mitochondrial-encoded peptides in avian species.