Skip to main content
SpringerLink
Log in

Investigating Mitochondrial Transcriptomes and RNA Processing Using Circular RNA Sequencing

  • Protocol
  • First Online:
Mitochondrial Gene Expression

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

Abstract

Transcriptomic technologies have revolutionized the study of gene expression and RNA biology. Different RNA sequencing methods enable the analyses of diverse species of transcripts, including their abundance, processing, stability, and other specific features. Mitochondrial transcriptomics has benefited from these technologies that have revealed the surprising complexity of its RNAs. Here we describe a method based upon cyclization of mitochondrial RNAs and next generation sequencing to analyze the steady-state levels and sizes of mitochondrial RNAs, their degradation products, as well as their processing intermediates by capturing both 5′ and 3′ ends of transcripts.

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. Mercer TR et al (2011) The human mitochondrial transcriptome. Cell 146:645–658

    Article  CAS  Google Scholar 

  2. Rackham O, Mercer TR, Filipovska A (2012) The human mitochondrial transcriptome and the RNA-binding proteins that regulate its expression. WIREs RNA 3:675–695

    Article  CAS  Google Scholar 

  3. Hallberg BM, Larsson N-G (2014) Making proteins in the powerhouse. Cell Metab 20:226–240

    Article  Google Scholar 

  4. Ferreira N, Rackham O, Filipovska A (2018) Regulation of a minimal transcriptome by repeat domain proteins. Semin Cell Dev Biol 76:132. https://doi.org/10.1016/j.semcdb.2017.08.037

    Article  CAS  PubMed  Google Scholar 

  5. Lee RG, Rudler DL, Rackham O, Filipovska A (2018) Is mitochondrial gene expression coordinated or stochastic? Biochem Soc Trans 46:1239–1246

    Article  Google Scholar 

  6. Rackham O et al (2011) Long noncoding RNAs are generated from the mitochondrial genome and regulated by nuclear-encoded proteins. RNA 17:2085–2093

    Article  CAS  Google Scholar 

  7. Montoya J, Ojala D, Attardi G (1981) Distinctive features of the 5′-terminal sequences of the human mitochondrial mRNAs. Nature 290:465–470

    Article  CAS  Google Scholar 

  8. Rackham O et al (2016) Hierarchical RNA processing is required for mitochondrial ribosome assembly. Cell Rep 16:1874–1890

    Article  CAS  Google Scholar 

  9. Perks KL et al (2018) PTCD1 is required for 16S rRNA maturation complex stability and mitochondrial ribosome assembly. Cell Rep 23:127–142

    Article  CAS  Google Scholar 

  10. Siira SJ et al (2018) Concerted regulation of mitochondrial and nuclear non-coding RNAs by a dual-targeted RNase Z. EMBO Rep 19:e46198. https://doi.org/10.15252/embr.201846198

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Siira SJ, Shearwood A-MJ, Bracken CP, Rackham O, Filipovska A (2017) Defects in RNA metabolism in mitochondrial disease. Int J Biochem Cell Biol 85:106–113. https://doi.org/10.1016/j.biocel.2017.02.003

    Article  CAS  PubMed  Google Scholar 

  12. Kühl I et al (2017) Transcriptomic and proteomic landscape of mitochondrial dysfunction reveals secondary coenzyme Q deficiency in mammals. elife 6:1494

    Article  Google Scholar 

  13. Gustafsson CM, Falkenberg M, Larsson N-G (2016) Maintenance and expression of mammalian mitochondrial DNA. Annu Rev Biochem 85:133–160

    Article  CAS  Google Scholar 

  14. Kuehl I et al (2016) POLRMT regulates the switch between replication primer formation and gene expression of mammalian mtDNA. Sci Adv 2:e1600963

    Article  Google Scholar 

  15. German MA et al (2008) Global identification of microRNA-target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol 26:941–946

    Google Scholar 

  16. Rackham O, Filipovska A (2014) Methods in molecular biology, vol 1125. Humana Press, Totowa, NJ, pp 263–275

    Google Scholar 

  17. Acevedo A, Brodsky L, Andino R (2015) Mutational and fitness landscapes of an RNA virus revealed through population sequencing. Nature 505:686–690

    Article  Google Scholar 

  18. Chu Y et al (2015) Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells. Nucleic Acids Res 43:e75–e75

    Article  Google Scholar 

  19. Kuznetsova I et al (2017) Simultaneous processing and degradation of mitochondrial RNAs revealed by circularized RNA sequencing. Nucleic Acids Res 5:5487–5500

    Article  Google Scholar 

  20. Wingett SW, Andrews S (2018) FastQ Screen: a tool for multi-genome mapping and quality control. F1000Res 7:1338

    Article  Google Scholar 

  21. Martin M (2011) Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J 17:10–12

    Article  Google Scholar 

  22. Magoč T, Salzberg SL (2011) FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27:2957–2963

    Article  Google Scholar 

  23. Gordon A (2010) Unpublished. G. H. F. A. S.-R. P. T. Fastx-toolkit

    Google Scholar 

  24. Benson G (1999) Tandem repeats finder: a program to analyze DNA sequences. Nucleic Acids Res 27:573–580

    Article  CAS  Google Scholar 

  25. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  Google Scholar 

  26. Krzywinski M et al (2009) Circos: An information aesthetic for comparative genomics. Genes Dev 19:1639–1645

    CAS  Google Scholar 

  27. Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Nat Commun 26:841–842

    CAS  Google Scholar 

  28. Ihaka R, Gentleman R (2012) R: a language for data analysis and graphics. J Comput Graph Stat 5:299–314

    Google Scholar 

  29. Rossum G (1995) Python reference manual. CWI, Amsterdam

    Google Scholar 

Download references

Acknowledgements

The work was supported by fellowships and project grants from the National Health and Medical Research Council (APP1159594, APP1154932, APP1154646 to AF and OR), Australian Research Council (to AF and OR), the Cancer Council of Western Australia (to OR and AF). IK is supported by a UWA Postgraduate Scholarship.

Author information

Authors and Affiliations

  1. Harry Perkins Institute of Medical Research, Nedlands, WA, Australia

    Irina Kuznetsova, Oliver Rackham & Aleksandra Filipovska

  2. ARC Centre of Excellence in Synthetic Biology, QEII Medical Centre, Nedlands, WA, Australia

    Irina Kuznetsova, Oliver Rackham & Aleksandra Filipovska

  3. Centre for Medical Research, The University of Western Australia, QEII Medical Centre, Nedlands, WA, Australia

    Irina Kuznetsova & Aleksandra Filipovska

  4. School of Pharmacy and Biomedical Sciences, Curtin University, Bentley, WA, Australia

    Oliver Rackham

  5. Curtin Health Innovation Research Institute, Curtin University, Bentley, WA, Australia

    Oliver Rackham

  6. Telethon Kids Institute, Northern Entrance, Perth Children’s Hospital, Nedlands, WA, Australia

    Oliver Rackham & Aleksandra Filipovska

  7. School of Molecular Sciences, The University of Western Australia, Crawley, WA, Australia

    Aleksandra Filipovska

Authors
  1. Irina Kuznetsova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Oliver Rackham
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Aleksandra Filipovska
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Aleksandra Filipovska .

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

Kuznetsova, I., Rackham, O., Filipovska, A. (2021). Investigating Mitochondrial Transcriptomes and RNA Processing Using Circular RNA Sequencing. 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_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0834-0_4

  • 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