The assembly of the mitoribosomal small subunit involves folding and modification of rRNA, and its association with mitoribosomal proteins. This process is assisted by a dynamic network of assembly factors. Conserved methyltransferases Mettl15 and Mettl17 act on the solvent-exposed surface of rRNA. Binding of Mettl17 is associated with the early assembly stage, whereas Mettl15 is involved in the late stage, but the mechanism of transition between the two was unclear. Here, we integrate structural data from Trypanosoma brucei with mammalian homologs and molecular dynamics simulations. We reveal how the interplay of Mettl15 and Mettl17 in intermediate steps links the distinct stages of small subunit assembly. The analysis suggests a model wherein Mettl17 acts as a platform for Mettl15 recruitment. Subsequent release of Mettl17 allows a conformational change of Mettl15 for substrate recognition. Upon methylation, Mettl15 adopts a loosely bound state which ultimately leads to its replacement by initiation factors, concluding the assembly. Together, our results indicate that assembly factors Mettl15 and Mettl17 cooperate to regulate the biogenesis process, and present a structural data resource for understanding molecular adaptations of assembly factors in mitoribosome.

Center for Theoretical Biological Physics Northeastern University Boston MA 02115 USA

Department of Integrated Structural Biology Institute of Genetics and Molecular and Cellular Biology University of Strasbourg Illkirch France

Department of Physics Northeastern University Boston MA 02115 USA

Department of Zoology Faculty of Science Charles University Prague Czech Republic

Dept of Cell and Molecular Biology National Bioinformatics Infrastructure Sweden Science for Life Laboratory Uppsala University Husargatan 3 SE 752 37 Uppsala Sweden

Dept of Physics Chemistry and Biology National Bioinformatics Infrastructure Sweden Science for Life Laboratory Linköping University 581 83 Linköping Sweden

Faculty of Science University of South Bohemia 37005 České Budějovice Czech Republic

Institute of Parasitology Biology Centre Czech Academy of Sciences 37005 České Budějovice Czech Republic

Science for Life Laboratory Department of Biochemistry and Biophysics Stockholm University 17165 Solna Sweden

See more in PubMed

Singh V. et al. Structural basis of LRPPRC-SLIRP-dependent translation by the mitoribosome. Nat Struct Mol Biol (2024). PubMed PMC

Itoh Y. et al. Mechanism of membrane-tethered mitochondrial protein synthesis. Science 371, 846–849 (2021). PubMed PMC

Ott M., Amunts A. & Brown A. Organization and Regulation of Mitochondrial Protein Synthesis. Annu Rev Biochem 85, 77–101 (2016). PubMed

Singh V. et al. Mitoribosome structure with cofactors and modifications reveals mechanism of ligand binding and interactions with L1 stalk. Nat Commun 15, 4272 (2024). PubMed PMC

Amunts A., Brown A., Toots J., Scheres S.H.W. & Ramakrishnan V. Ribosome. The structure of the human mitochondrial ribosome. Science 348, 95–98 (2015). PubMed PMC

Greber B.J. et al. Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348, 303–8 (2015). PubMed

Lavdovskaia E. et al. A roadmap for ribosome assembly in human mitochondria. Nat Struct Mol Biol (2024). PubMed PMC

Conor Moran J., Del’Olio S., Choi A., Zhong H. & Barrientos A. Mitoribosome Biogenesis. Methods Mol Biol 2661, 23–51 (2023). PubMed PMC

Brischigliaro M., Sierra-Magro A., Ahn A. & Barrientos A. Mitochondrial ribosome biogenesis and redox sensing. FEBS Open Bio 14, 1640–1655 (2024). PubMed PMC

Khawaja A., Cipullo M., Kruger A. & Rorbach J. Insights into mitoribosomal biogenesis from recent structural studies. Trends Biochem Sci (2023). PubMed

Haas R.H. Mitochondrial Dysfunction in Aging and Diseases of Aging. Biology (Basel) 8(2019). PubMed PMC

Hong H.J. et al. Mitoribosome insufficiency in beta cells is associated with type 2 diabetes-like islet failure. Exp Mol Med 54, 932–945 (2022). PubMed PMC

Richman T.R. et al. Mitochondrial mistranslation modulated by metabolic stress causes cardiovascular disease and reduced lifespan. Aging Cell 20, e13408 (2021). PubMed PMC

Pecina P. et al. Haplotype variability in mitochondrial rRNA predisposes to metabolic syndrome. Commun Biol 7, 1116 (2024). PubMed PMC

Saurer M. et al. Mitoribosomal small subunit biogenesis in trypanosomes involves an extensive assembly machinery. Science 365, 1144–1149 (2019). PubMed

Soufari H. et al. Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis. Proc Natl Acad Sci U S A 117, 29851–29861 (2020). PubMed PMC

Jaskolowski M. et al. Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis. Mol Cell 79, 629–644 e4 (2020). PubMed

Tobiasson V. et al. Interconnected assembly factors regulate the biogenesis of mitoribosomal large subunit. EMBO J 40, e106292 (2021). PubMed PMC

Lenarcic T. et al. Mitoribosomal small subunit maturation involves formation of initiation-like complexes. Proc Natl Acad Sci U S A 119(2022). PubMed PMC

Kummer E. et al. Unique features of mammalian mitochondrial translation initiation revealed by cryo-EM. Nature 560, 263–267 (2018). PubMed

Khawaja A. et al. Distinct pre-initiation steps in human mitochondrial translation. Nat Commun 11, 2932 (2020). PubMed PMC

Itoh Y. et al. Mechanism of mitoribosomal small subunit biogenesis and preinitiation. Nature (2022). PubMed PMC

Itoh Y. et al. Structure of the mitoribosomal small subunit with streptomycin reveals Fe-S clusters and physiological molecules. Elife 11(2022). PubMed PMC

Aibara S., Singh V., Modelska A. & Amunts A. Structural basis of mitochondrial translation. Elife 9(2020). PubMed PMC

Pham T.C.P. et al. The mitochondrial mRNA-stabilizing protein SLIRP regulates skeletal muscle mitochondrial structure and respiration by exercise-recoverable mechanisms. Nat Commun 15, 9826 (2024). PubMed PMC

Harper N.J., Burnside C. & Klinge S. Principles of mitoribosomal small subunit assembly in eukaryotes. Nature 614, 175–181 (2023). PubMed PMC

Ast T. et al. METTL17 is an Fe-S cluster checkpoint for mitochondrial translation. Mol Cell 84, 359–374 e8 (2024). PubMed PMC

Evans R. et al. (2022).

Wallner B., Amunts A., Naschberger A., Nystedt B. & Mirabello C. (2022).

Jumper J. & Hassabis D. Protein structure predictions to atomic accuracy with AlphaFold. Nat Methods 19, 11–12 (2022). PubMed

Schedlbauer A. et al. A conserved rRNA switch is central to decoding site maturation on the small ribosomal subunit. Sci Adv 7(2021). PubMed PMC

Bikmullin A.G. et al. Yet Another Similarity between Mitochondrial and Bacterial Ribosomal Small Subunit Biogenesis Obtained by Structural Characterization of RbfA from S. aureus. International Journal of Molecular Sciences 24(2023). PubMed PMC

Datta P.P. et al. Structural aspects of RbfA action during small ribosomal subunit assembly. Mol Cell 28, 434–45 (2007). PubMed PMC

Rozanska A. et al. The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome. Biochem J 474, 2145–2158 (2017). PubMed PMC

Van Haute L. et al. METTL15 introduces N4-methylcytidine into human mitochondrial 12S rRNA and is required for mitoribosome biogenesis. Nucleic Acids Res 47, 10267–10281 (2019). PubMed PMC

Laptev I. et al. METTL15 interacts with the assembly intermediate of murine mitochondrial small ribosomal subunit to form m4C840 12S rRNA residue. Nucleic Acids Res 48, 8022–8034 (2020). PubMed PMC

Mutti C.D., Van Haute L. & Minczuk M. The catalytic activity of methyltransferase METTL15 is dispensable for its role in mitochondrial ribosome biogenesis. RNA Biol 21, 23–30 (2024). PubMed PMC

Chen H. et al. The human mitochondrial 12S rRNA m(4)C methyltransferase METTL15 is required for mitochondrial function. J Biol Chem 295, 8505–8513 (2020). PubMed PMC

Shi Z. et al. Mettl17, a regulator of mitochondrial ribosomal RNA modifications, is required for the translation of mitochondrial coding genes. FASEB J 33, 13040–13050 (2019). PubMed

Mashkovskaia A.V. et al. Testing a Hypothesis of 12S rRNA Methylation by Putative METTL17 Methyltransferase. Acta Naturae 15, 75–82 (2023). PubMed PMC

Zhong H. et al. BOLA3 and NFU1 link mitoribosome iron-sulfur cluster assembly to multiple mitochondrial dysfunctions syndrome. Nucleic Acids Res 51, 11797–11812 (2023). PubMed PMC

Gahura O., Chauhan P. & Zikova A. Mechanisms and players of mitoribosomal biogenesis revealed in trypanosomatids. Trends Parasitol 38, 1053–1067 (2022). PubMed

Tyc J., Novotna L., Pena-Diaz P., Maslov D.A. & Lukes J. RSM22, mtYsxC and PNKD-like proteins are required for mitochondrial translation in Trypanosoma brucei. Mitochondrion 34, 67–74 (2017). PubMed

Dimmer K.S. et al. Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae. Mol Biol Cell 13, 847–53 (2002). PubMed PMC

Petrov A.S. et al. Structural Patching Fosters Divergence of Mitochondrial Ribosomes. Mol Biol Evol 36, 207–219 (2019). PubMed PMC

Zhu W., Shenoy A., Kundrotas P. & Elofsson A. Evaluation of AlphaFold-Multimer prediction on multi-chain protein complexes. Bioinformatics 39(2023). PubMed PMC

Case David A., et al. “The Amber biomolecular simulation programs.” Journal of computational chemistry 26.16 (2005): 1668–1688. PubMed PMC

Noel J.K. et al. SMOG 2: A Versatile Software Package for Generating Structure-Based Models. PLoS Comput Biol 12, e1004794 (2016). PubMed PMC

Whitford P.C. et al. Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways. RNA 16, 1196–204 (2010). PubMed PMC

Jackson J., Nguyen K. & Whitford P.C. Exploring the balance between folding and functional dynamics in proteins and RNA. Int J Mol Sci 16, 6868–89 (2015). PubMed PMC

Freitas F.C., Fuchs G., de Oliveira R.J. & Whitford P.C. The dynamics of subunit rotation in a eukaryotic ribosome. Biophysica 1, 204–221 (2021). PubMed PMC

Averina O.A. et al. Mitochondrial rRNA Methylation by Mettl15 Contributes to the Exercise and Learning Capability in Mice. Int J Mol Sci 23(2022). PubMed PMC

Glasgow R.I.C. et al. The mitochondrial methylation potential gates mitoribosome assembly. in bioRxiv (2024).

Li H., Yu K., Hu H., Zhang X., Zeng S., Li J., … & Zhang Y. METTL17 coordinates ferroptosis and tumorigenesis by regulating mitochondrial translation in colorectal cancer. Redox Biology, 71, 103087 (2024). PubMed PMC

Jamali K. et al. Automated model building and protein identification in cryo-EM maps. Nature 628, 450–457 (2024). PubMed PMC

Su B., Huang K., Peng Z., Amunts A. & Yang J. Improved automated model building for cryo-EM maps using CryFold. in bioRxiv (2024).

Wang X., Zhu H., Terashi G., Taluja M. & Kihara D. DiffModeler: large macromolecular structure modeling for cryo-EM maps using a diffusion model. Nat Methods 21, 2307–2317 (2024). PubMed

Lewis S. et al. (2024).

Pu Y.G. et al. Crystal structures and putative interface of Saccharomyces cerevisiae mitochondrial matrix proteins Mmf1 and Mam33. J Struct Biol 175, 469–74 (2011). PubMed

Jiang J., Zhang Y., Krainer A.R. & Xu R.M. Crystal structure of human p32, a doughnut-shaped acidic mitochondrial matrix protein. Proc Natl Acad Sci U S A 96, 3572–7 (1999). PubMed PMC

Waltz F. et al. How to build a ribosome from RNA fragments in Chlamydomonas mitochondria. Nat Commun 12, 7176 (2021). PubMed PMC

Burki F., Roger A.J., Brown M.W. & Simpson A.G.B. The New Tree of Eukaryotes. Trends Ecol Evol 35, 43–55 (2020). PubMed

Krissinel E. & Henrick K. Inference of macromolecular assemblies from crystalline state. J Mol Biol 372, 774–97 (2007). PubMed

Mirabello C., Wallner B., Nystedt B., Azinas S., & Carroni M. Unmasking AlphaFold to integrate experiments and predictions in multimeric complexes. NatureCommunications, 15(1), 8724 (2024). PubMed PMC

Schrödinger L. & DeLano W., 2020. PyMOL, Available at: http://www.pymol.org/pymol.

Richter D.J. et al. EukProt: A database of genome-scale predicted proteins across the diversity of eukaryotes. Peer Community Journal 2(2022).

Mistry J., Finn R.D., Eddy S.R., Bateman A. & Punta M. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res 41, e121 (2013). PubMed PMC

Altschul S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–402 (1997). PubMed PMC

Katoh K. & Standley D.M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30, 772–80 (2013). PubMed PMC

Nguyen L.T., Schmidt H.A., von Haeseler A. & Minh B.Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol 32, 268–74 (2015). PubMed PMC

Hassan A. et al. Ratchet, swivel, tilt and roll: a complete description of subunit rotation in the ribosome. Nucleic Acids Res 51, 919–934 (2023). PubMed PMC

Nguyen K. & Whitford P.C. Steric interactions lead to collective tilting motion in the ribosome during mRNA-tRNA translocation. Nat Commun 7, 10586 (2016). PubMed PMC

de Oliveira A.B. Jr. et al. SMOG 2 and OpenSMOG: Extending the limits of structure-based models. Protein Sci 31, 158–172 (2022). PubMed PMC

Eastman P. et al. OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials. J Phys Chem B 128, 109–116 (2024). PubMed PMC

Crooks G.E., Hon G., Chandonia J.M. & Brenner S.E. WebLogo: a sequence logo generator. Genome Res 14, 1188–90 (2004). PubMed PMC

Yariv B. et al. Using evolutionary data to make sense of macromolecules with a “face-lifted” ConSurf. Protein Sci 32, e4582 (2023). PubMed PMC