Skip to main content
SpringerLink
Log in

Mass Spectrometric Analysis of Mitochondrial RNA Modifications

  • Protocol
  • First Online:
Mitochondrial Gene Expression

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2192))

Abstract

Mitochondrial RNAs are modified posttranscriptionally. These modifications are required for proper functioning of RNA molecules, and thereby contribute to essential mitochondrial processes. Herein, we describe our latest mass spectrometry-based platform for analysis of posttranscriptional modifications of mitochondrial tRNAs, and measuring the in vitro activity of mitochondrial RNA-modifying enzymes.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Barrell BG, Bankier AT, Drouin J (1979) A different genetic code in human mitochondria. Nature 282(5735):189–194. https://doi.org/10.1038/282189a0

    Article  CAS  PubMed  Google Scholar 

  2. Suzuki T, Nagao A, Suzuki T (2011) Human mitochondrial diseases caused by lack of taurine modification in mitochondrial tRNAs. Wiley Interdiscip Rev RNA 2(3):376–386. https://doi.org/10.1002/wrna.65

    Article  CAS  PubMed  Google Scholar 

  3. Hallberg BM, Larsson NG (2014) Making proteins in the powerhouse. Cell Metab 20(2):226–240. https://doi.org/10.1016/j.cmet.2014.07.001

    Article  CAS  PubMed  Google Scholar 

  4. Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290(5806):470–474. https://doi.org/10.1038/290470a0

    Article  CAS  PubMed  Google Scholar 

  5. Rebelo-Guiomar P, Powell CA, Van Haute L, Minczuk M (2019) The mammalian mitochondrial epitranscriptome. Biochim Biophys Acta Gene Regul Mech. 1862(3):429–446. https://doi.org/10.1016/j.bbagrm.2018.11.005

  6. Safra M, Sas-Chen A, Nir R, Winkler R, Nachshon A, Bar-Yaacov D, Erlacher M, Rossmanith W, Stern-Ginossar N, Schwartz S (2017) The m1A landscape on cytosolic and mitochondrial mRNA at single-base resolution. Nature 551(7679):251–255. https://doi.org/10.1038/nature24456

    Article  CAS  PubMed  Google Scholar 

  7. Suzuki T (2005) Biosynthesis and function of tRNA Wobble modifications. In: Grosjean H (ed) Fine-tuning of RNA functions by modification and editing, Topics in current genetics, vol 12. Springer, Berlin, pp 23–69

    Chapter  Google Scholar 

  8. Boccaletto P, Machnicka MA, Purta E, Piatkowski P, Baginski B, Wirecki TK, de Crecy-Lagard V, Ross R, Limbach PA, Kotter A, Helm M, Bujnicki JM (2018) MODOMICS: a database of RNA modification pathways. 2017 update. Nucleic Acids Res 46(D1):D303–D307. https://doi.org/10.1093/nar/gkx1030

    Article  CAS  PubMed  Google Scholar 

  9. Asano K, Suzuki T, Saito A, Wei FY, Ikeuchi Y, Numata T, Tanaka R, Yamane Y, Yamamoto T, Goto T, Kishita Y, Murayama K, Ohtake A, Okazaki Y, Tomizawa K, Sakaguchi Y, Suzuki T (2018) Metabolic and chemical regulation of tRNA modification associated with taurine deficiency and human disease. Nucleic Acids Res 46(4):1565–1583. https://doi.org/10.1093/nar/gky068

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Suzuki T, Suzuki T (2007) Chaplet column chromatography: isolation of a large set of individual RNAs in a single step. Methods Enzymol 425:231–239. https://doi.org/10.1016/S0076-6879(07)25010-4

    Article  CAS  PubMed  Google Scholar 

  11. Suzuki T, Suzuki T, Wada T, Saigo K, Watanabe K (2002) Taurine as a constituent of mitochondrial tRNAs: new insights into the functions of taurine and human mitochondrial diseases. EMBO J 21(23):6581–6589. https://doi.org/10.1093/emboj/cdf656

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Miyauchi K, Ohara T, Suzuki T (2007) Automated parallel isolation of multiple species of non-coding RNAs by the reciprocal circulating chromatography method. Nucleic Acids Res 35(4):e24. https://doi.org/10.1093/nar/gkl1129

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Sakaguchi Y, Miyauchi K, Kang BI, Suzuki T (2015) Nucleoside analysis by hydrophilic interaction liquid chromatography coupled with mass spectrometry. Methods Enzymol 560:19–28. https://doi.org/10.1016/bs.mie.2015.03.015

    Article  CAS  PubMed  Google Scholar 

  14. Suzuki T, Suzuki T (2014) A complete landscape of post-transcriptional modifications in mammalian mitochondrial tRNAs. Nucleic Acids Res 42(11):7346–7357. https://doi.org/10.1093/nar/gku390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Chujo T, Suzuki T (2012) Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs. RNA 18(12):2269–2276. https://doi.org/10.1261/rna.035600.112

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bar-Yaacov D, Frumkin I, Yashiro Y, Chujo T, Ishigami Y, Chemla Y, Blumberg A, Schlesinger O, Bieri P, Greber B, Ban N, Zarivach R, Alfonta L, Pilpel Y, Suzuki T, Mishmar D (2016) Mitochondrial 16S rRNA is methylated by tRNA methyltransferase TRMT61B in all vertebrates. PLoS Biol 14(9):e1002557. https://doi.org/10.1371/journal.pbio.1002557

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ohira T, Suzuki T (2016) Precursors of tRNAs are stabilized by methylguanosine cap structures. Nat Chem Biol 12(8):648–655. https://doi.org/10.1038/nchembio.2117

    Article  CAS  PubMed  Google Scholar 

  18. Mengel-Jorgensen J, Kirpekar F (2002) Detection of pseudouridine and other modifications in tRNA by cyanoethylation and MALDI mass spectrometry. Nucleic Acids Res 30(23):e135. https://doi.org/10.1093/nar/gnf135

    Article  PubMed  PubMed Central  Google Scholar 

  19. Cantara WA, Crain PF, Rozenski J, McCloskey JA, Harris KA, Zhang X, Vendeix FA, Fabris D, Agris PF (2011) The RNA modification database, RNAMDB: 2011 update. Nucleic Acids Res 39(Database issue):D195–D201. https://doi.org/10.1093/nar/gkq1028

  20. Yu N, Lobue PA, Cao X, Limbach PA (2017) RNAModMapper: RNA modification mapping software for analysis of liquid chromatography tandem mass spectrometry data. Anal Chem 89(20):10744–10752. https://doi.org/10.1021/acs.analchem.7b01780

    Article  CAS  PubMed  Google Scholar 

  21. Lin H, Miyauchi K, Harada T, Okita R, Takeshita E, Komaki H, Fujioka K, Yagasaki H, Goto YI, Yanaka K, Nakagawa S, Sakaguchi Y, Suzuki T (2018) CO2-sensitive tRNA modification associated with human mitochondrial disease. Nat Commun 9(1):1875. https://doi.org/10.1038/s41467-018-04250-4

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Juhling F, Morl M, Hartmann RK, Sprinzl M, Stadler PF, Putz J (2009) tRNAdb 2009: compilation of tRNA sequences and tRNA genes. Nucleic Acids Res 37(Database issue):D159–162F. https://doi.org/10.1093/nar/gkn772

    Article  CAS  PubMed  Google Scholar 

  23. Suzuki T, Yashiro Y, Kikuchi I, Ishigami Y, Saito H, Matsuzawa I, Okada S, Mito M, Iwasaki S, Ma D, Zhao X, Asano K, Lin H, Kirino Y, Sakaguchi Y, Suzuki T (2020) Complete chemical structures of human mitochondrial tRNAs. Nat Commun in press

    Google Scholar 

Download references

Acknowledgments

We thank Yuriko Sakaguchi and Yuka Yashiro for technical support, and Dr. Yohei Kirono (Thomas Jefferson University) for providing materials. This work was funded by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), and the Japan Society for the Promotion of Science (JSPS; grant no. 26702035 and 18H02094 to Ta. S., and 26113003, 26220205, and 18H05272 to Ts. S.).

Author information

Authors and Affiliations

  1. Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Tokyo, Japan

    Yuma Ishigami, Tsutomu Suzuki & Takeo Suzuki

Authors
  1. Yuma Ishigami
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tsutomu Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takeo Suzuki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Tsutomu Suzuki or Takeo Suzuki .

Editor information

Editors and Affiliations

  1. Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK

    Michal Minczuk

  2. Division of Molecular Metabolism, Department of Medical Biochemistry and Biophysics, Karolinska Institute, Solna, Sweden, Institutet Laboratory, Max Planck Institute Biology of Ageing - Karolinska, Karolinska Institutet, Stockholm, Sweden

    Joanna Rorbach

Rights and permissions

Reprints and permissions

Copyright information

© 2021 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Ishigami, Y., Suzuki, T., Suzuki, T. (2021). Mass Spectrometric Analysis of Mitochondrial RNA Modifications. In: Minczuk, M., Rorbach, J. (eds) Mitochondrial Gene Expression. Methods in Molecular Biology, vol 2192. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0834-0_8

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0834-0_8

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0833-3

  • Online ISBN: 978-1-0716-0834-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation