Abstract
Like bacterial and cytoplasmic ribosomes, mitoribosomes are large ribonucleoprotein complexes with molecular weights in the range of several million Daltons. Traditionally, studying the assembly of such high molecular weight complexes is done using ultracentrifugation through linear density gradients, which remains the method of choice due to its versatility and superior resolving power in the high molecular weight range. Here, we present a protocol for the analysis of mitoribosomal assembly in heart mitochondrial extracts using linear density sucrose gradients that we have previously employed to characterize the essential role of different mitochondrial proteins in mitoribosomal biogenesis. This protocol details in a stepwise manner a typical mitoribosomal assembly analysis starting with isolation of mitochondria, preparation and ultracentrifugation of the gradients, fractionation and ending with SDS-PAGE, and immunoblotting of the gradient fractions. Even though we provide an example with heart mitochondria, this protocol can be directly applied to virtually all mouse tissues, as well as cultured cells, with little to no modifications.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Brown A, Amunts A, Bai XC, Sugimoto Y et al (2014) Structure of the large ribosomal subunit from human mitochondria. Science 346(6210):718–722. https://doi.org/10.1126/science.1258026
Greber BJ, Boehringer D, Leibundgut M, Bieri P et al (2014) The complete structure of the large subunit of the mammalian mitochondrial ribosome. Nature 515(7526):283–286. https://doi.org/10.1038/nature13895
Kaushal PS, Sharma MR, Booth TM, Haque EM et al (2014) Cryo-EM structure of the small subunit of the mammalian mitochondrial ribosome. Proc Natl Acad Sci U S A 111(20):7284–7289. https://doi.org/10.1073/pnas.1401657111
Amunts A, Brown A, Toots J, Scheres SH et al (2015) Ribosome. The structure of the human mitochondrial ribosome. Science 348(6230):95–98. https://doi.org/10.1126/science.aaa1193
Greber BJ, Bieri P, Leibundgut M, Leitner A et al (2015) Ribosome. The complete structure of the 55S mammalian mitochondrial ribosome. Science 348(6232):303–308. https://doi.org/10.1126/science.aaa3872
Rorbach J, Gao F, Powell CA, D’Souza A et al (2016) Human mitochondrial ribosomes can switch their structural RNA composition. Proc Natl Acad Sci U S A 113(43):12198–12201. https://doi.org/10.1073/pnas.1609338113
Chrzanowska-Lightowlers Z, Rorbach J, Minczuk M (2017) Human mitochondrial ribosomes can switch structural tRNAs - but when and why? RNA Biol 14(12):1668–1671. https://doi.org/10.1080/15476286.2017.1356551
Metodiev MD, Lesko N, Park CB, Camara Y et al (2009) Methylation of 12S rRNA is necessary for in vivo stability of the small subunit of the mammalian mitochondrial ribosome. Cell Metab 9(4):386–397. https://doi.org/10.1016/j.cmet.2009.03.001
Metodiev MD, Spahr H, Loguercio Polosa P, Meharg C et al (2014) NSUN4 is a dual function mitochondrial protein required for both methylation of 12S rRNA and coordination of mitoribosomal assembly. PLoS Genet 10(2):e1004110. https://doi.org/10.1371/journal.pgen.1004110
Camara Y, Asin-Cayuela J, Park CB, Metodiev MB et al (2011) MTERF4 regulates translation by targeting the methyltransferase NSUN4 to the mammalian mitochondrial ribosome. Cell Metab 13(5):527–539. https://doi.org/10.1016/j.cmet.2011.04.002
Wredenberg A, Lagouge M, Bratic A, Metodiev MD et al (2013) MTERF3 regulates mitochondrial ribosome biogenesis in invertebrates and mammals. PLoS Genet 9(1):e1003178. https://doi.org/10.1371/journal.pgen.1003178
Bruning JC, Michael MD, Winnay JN, Hayashi T et al (1998) A muscle-specific insulin receptor knockout exhibits features of the metabolic syndrome of NIDDM without altering glucose tolerance. Mol Cell 2(5):559–569
Sakamoto K, Gurumurthy CB, Wagner KU (2014) Generation of conditional knockout mice. Methods Mol Biol 1194:21–35. https://doi.org/10.1007/978-1-4939-1215-5_2
Cahill A, Baio DL, Cunningham CC (1995) Isolation and characterization of rat liver mitochondrial ribosomes. Anal Biochem 232(1):47–55. https://doi.org/10.1006/abio.1995.9962
O’Brien TW (1971) The general occurrence of 55 S ribosomes in mammalian liver mitochondria. J Biol Chem 246(10):3409–3417
Hamilton MG, O’Brien TW (1974) Ultracentrifugal characterization of the mitochondrial ribosome and subribosomal particles of bovine liver: molecular size and composition. Biochemistry 13(26):5400–5403. https://doi.org/10.1021/bi00723a024
Ruzzenente B, Metodiev MD, Wredenberg A, Bratic A et al (2012) LRPPRC is necessary for polyadenylation and coordination of translation of mitochondrial mRNAs. EMBO J 31(2):443–456. https://doi.org/10.1038/emboj.2011.392
Luthe DS (1983) A simple technique for the preparation and storage of sucrose gradients. Anal Biochem 135(1):230–232
Cooper AJ, Smallwood JA, Morgan RA (1984) The preparation of freeze-thaw density gradients with homogeneous solute concentrations. J Immunol Methods 71(2):259–264
Lake NJ, Webb BD, Stroud DA, Richman TR et al (2017) Biallelic mutations in MRPS34 lead to instability of the small mitoribosomal subunit and leigh syndrome. Am J Hum Genet 101(2):239–254. https://doi.org/10.1016/j.ajhg.2017.07.005
Lambowitz AM (1979) Preparation and analysis of mitochondrial ribosomes. Methods Enzymol 59:421–433
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Ruzzenente, B., Metodiev, M.D. (2021). Linear Density Sucrose Gradients to Study Mitoribosomal Biogenesis in Tissue-Specific Knockout Mice. In: Singh, S.R., Hoffman, R.M., Singh, A. (eds) Mouse Genetics . Methods in Molecular Biology, vol 2224. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1008-4_3
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1008-4_3
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1007-7
Online ISBN: 978-1-0716-1008-4
eBook Packages: Springer Protocols