Mitochondrial rRNA Methylation by Mettl15 Contributes to the Exercise and Learning Capability in Mice

Abstract

Mitochondrial translation is a unique relic of the symbiotic origin of the organelle. Alterations of its components cause a number of severe human diseases. Hereby we report a study of mice devoid of Mettl15 mitochondrial 12S rRNA methyltransferase, responsible for the formation of m4C839 residue (human numbering). Homozygous Mettl15−/− mice appeared to be viable in contrast to other mitochondrial rRNA methyltransferase knockouts reported earlier. The phenotype of Mettl15−/− mice is much milder than that of other mutants of mitochondrial translation apparatus. In agreement with the results obtained earlier for cell cultures with an inactivated Mettl15 gene, we observed accumulation of the RbfA factor, normally associated with the precursor of the 28S subunit, in the 55S mitochondrial ribosome fraction of knockout mice. A lack of Mettl15 leads to a lower blood glucose level after physical exercise relative to that of the wild-type mice. Mettl15−/− mice demonstrated suboptimal muscle performance and lower levels of Cox3 protein synthesized by mitoribosomes in the oxidative soleus muscles. Additionally, we detected decreased learning capabilities in the Mettl15−/− knockout mice in the tests with both positive and negative reinforcement. Such properties make Mettl15−/− knockout mice a suitable model for mild mitochondriopathies.

Structural insights into the specific recognition of mitochondrial ribosome-binding factor hsRBFA and 12 S rRNA by methyltransferase METTL15

“…Lactylation highlights the novel role of lactate in regulating transcription, cellular functions and disease pathogenesis 39 . Intriguingly, homozygous Mettl15 KO mice survived and exhibited a much milder phenotype than Tfb1m −/− mice, yet decreased exercise and learning capability were detected 40 . Furthermore, H3K9-la was recently identified to promote tumorigenicity of liver cancer stem 41 .…”

The catalytic activity of methyltransferase METTL15 is dispensable for its role in mitochondrial ribosome biogenesis

“…Inactivation of METTL15 has been shown in several models to impact mitochondrial function and ribosome biogenesis, with the severity substantially varying across these models [ 17–19 , 23 ]. In order to validate the previous work on the essential role of METTL15, we generated a knockout of the human METTL15 gene locus in HEK293T cells using CRISPR/Cas9 guides targeted to exon 3 (the first coding exon) (Supplementary Figure S1).…”

Kidney-Related Function of Mitochondrial Protein Mitoregulin

“…To stain beta-galactosidase-positive senescent cells, we used the protocol for paraffin sections created by V.N. Manskikh [39]; kidney samples were fixed with Baker's formolcalcium solution (1 g CaCl 2 , 10 mL 37% formaldehyde, 90 mL water) [40] during 24 h at +4 • C, washed in distilled water for 24 h, dehydrated in 3 changes of absolute acetone at +4 • C for 2 h in each, cleared 1 h in benzene and embedded in paraffin with Z. Lojda et al method for enzyme histochemistry [41]. Microtome sections with a thickness of 5 µm were stained 3 h with X-gal solution (20 mg X-gal, 1 mL Dimethylformamide, 5 mL 1.65% K 3 [Fe(CN) 6 ], 5 mL 2.11% K 4 [Fe(CN) 6 ], 70 mL 0.1 M citrate buffer pH 6.0) at suboptimal pH 6.0 and 37 • C as recommended [42] and counterstained with nuclear red.…”

“…Immunoblotting of tissue lysates was done as previously described [39]. For immunoblot analysis, samples of kidney tissue were lysed in RIPA buffer (150 mM sodium chloride, 50 mM Tris-HCl pH 8.0, 0.5% Nonidet P-40, 1% sodium deoxycholate, 0.5% SDS) with protease inhibitor cocktail (ThermoFisher Scientific, Waltham, MA, USA).…”

“…Assessment of the mitochondrial-to-nuclear DNA ratio was done as previously described [39]. DNA was extracted from 5-15 mg of kidney tissue with GeneJET Genomic DNA Purification Kit (ThermoFisher Scientific, Waltham, MA, USA).…”