The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress

I’m not a big believer in mitochondrial derived peptides. One of them is known as MOTS-c (mitochondrial open reading frame of the 12S rRNA-c) which is claimed to be transcribed within mitochondria from the 12S rRNA gene, exported into the cytosol and translated by the cytosolic ribosomes (Lee et al. 2015). In the case MOTS-c actually existed, it would not even be that conserved. I scraped the human 12S rDNA variants in the MOTS-c region from the mitomap database (https://www.mitomap.org/foswiki/bin/view/MITOMAP/PolymorphismsCoding accessed 09.07.2018). Simple alignment with Clustal omega shows that some non-pathogenic  human variants can remove the start codon, cause multiple nonsynonymous mutations and even remove the stop codon.

All known human variants listed in the MitoMap database and covering the MOTS-c region of mitochondrial DNA. On the left the type and position of mutation and on the right its effect (if any) on the predicted MOTS-c.

In addition, I don’t believe the current literature has excluded the possibility that MOTS-c could originate from a nuclear fragment of mtDNA (NUMT). For instance, a quick blast search identifies several positions in the human genome, where a NUMT could be used to express a MOTS-c or a MOTS-c-like peptides.

Blastn search using the MOTS-c sequence 5'-ATGAGGTGGCAAGAAATGGGCTACATTTTCTACCCCAGAAAACTACGATAG-3' against Human genomic plus transcript (Human H+T) database. Chromosome|Position|Predicted peptide. GRCh38.p12 is the reference sequence.

Now a new paper from Kim et al. suggests that MOTS-c is somehow involved in nuclear gene expression (Kim et al. 2018). In this manuscript they made a new antibody detecting the endogenous MOTS-c but didn’t describe at all how this was done. The last author of this manuscript is also the first author of the older MOTS-c publication (Lee et al. 2015) so it is weird why they didn’t use the same antibody. They used peptide competition to verify the specificity of their new antibody but it would have also been nice to see whether they detect something in ρ0 cells (cells lacking mtDNA).

In any case, in their subcellular fractionations MOTS-c seems to localize with mitochondria (Fig. 1A) although it is not clear whether it actually is within mitochondria or just stick to the outer membrane. In contrast, using immunocytochemistry to detect the endogenous peptide or over-expressed fluorophore tagged MOTS-c, the peptide shows a poor mitochondrial co-localization (Fig. 1B,D). They also tried to assess the subcellular localization of MOTS-c by combining it with enhanced green fluorescent protein (eGFP). To me it seems rather uninformative to tag a 16 amino acid peptide (MOTS-c) with a 239 amino eGFP and think this would have some biological relevance.

Both the MOTS-c eGPF and MOTS-c eGFP variants, where multiple residues are replaced with alanine, showed mitochondrial localization using subcellular fractionations (Fig. 1F). Interestingly over-expressing the MOTS-c variants, where 4 different amino acids are replaced with alanine (8YIFY11 > 8AAAA11, 13RKLR16 > 13AAAA16), had similar or even stronger effects on mitochondrial respiration (Fig. S1) suggesting to me they are hunting for over-expression artifacts. In their statistics they carried out multiple comparisons using Student’s t-test so it is hard to tell which differences were actually significant. In addition, it would have been interesting to see as a control, whether over-expressing a 16 amino acid long repeat of alanines or a scrambled peptide would have the same effect on mitochondrial respiration.

For the rest of the manuscript the authors mostly over-expressed MOTS-c and its variants in cells and observed all kinds of changes. Importantly, they didn’t really have a pure negative control in the form of an unrelated peptide and in every bar graph (Fig 2, 4, S1, S3, S4) they carried out multiple comparisons using Student’s t-test. All in all, this publication doesn’t make me any more convinced that MOTS-c is originating from mitochondria or that it has some biological relevance as most experiments are based on overexpression.

PS: The authors should note that only 11 of the mtDNA encoded proteins are part of the electron transport system and all 13 are part of the oxidative phosphorylation system.

References:

Kim KH, Son JM, Benayoun BA, Lee C. The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress. Cell Met. 2018. PMID:Not available

Lee C, Zeng J, Drew BG, Sallam T, Martin-Montalvo A, Wan J, Kim SJ, Mehta H, Hevener AL, de Cabo R, Cohen P. The mitochondrial-derived peptide MOTS-c promotes metabolic homeostasis and reduces obesity and insulin resistance. Cell Metab. 2015. PMID: 25738459