Skip to main content

Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes

  • Protocol
  • First Online:
Book cover Environmental Responses in Plants

Abstract

Posttranscriptional control makes an important contribution to circadian regulation of gene expression. In higher plants, alternative splicing is particularly prevalent upon abiotic and biotic stress and in the circadian system. Here we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) to monitor alternative splicing events. The use of the panel allows the quantification of changes in the proportion of splice isoforms between different samples, e.g., different time points, different tissues, genotypes, ecotypes, or treatments.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 129.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 179.99
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. Reddy ASN, Marquez Y, Kalyna M et al (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25:3657–3683

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  2. Staiger D, Brown JWS (2013) Alternative splicing at the intersection of biological timing, development, and stress responses. Plant Cell 25:3640–3656

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Streitner C, Hennig L, Korneli C et al (2010) Global transcript profiling of transgenic plants constitutively overexpressing the RNA-binding protein AtGRP7. BMC Plant Biol 10:221

    Article  PubMed Central  PubMed  Google Scholar 

  4. Hennig L, Menges M, Murray JA et al (2003) Arabidopsis transcript profiling on Affymetrix GeneChip arrays. Plant Mol Biol 53:457–465

    Article  CAS  PubMed  Google Scholar 

  5. Hazen SP, Naef F, Quisel T et al (2009) Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays. Genome Biol. 10: R17.

    Google Scholar 

  6. Simpson CG, Fuller J, Maronova M et al (2008) Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts. Plant J 53:1035–1048

    Article  CAS  PubMed  Google Scholar 

  7. Raczynska KD, Simpson CG, Ciesiolka A et al (2010) Involvement of the nuclear cap-binding protein complex in alternative splicing in Arabidopsis thaliana. Nucleic Acids Res 38:265–278

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  8. Streitner C, Köster T, Simpson CG et al (2012) An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with target transcripts in Arabidopsis thaliana. Nucleic Acids Res 40:11240–11255

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Streitner C, Simpson CG, Shaw P et al (2013) Small changes in ambient temperature affect alternative splicing in Arabidopsis thaliana. Plant Signal Behav 8:e24638

    Article  PubMed Central  PubMed  Google Scholar 

  10. Filichkin SA, Priest HD, Givan SA et al (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20:45–58

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Marquez Y, Brown JWS, Simpson CG et al (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22:1184–1195

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  12. Staiger D, Shin J, Johansson M et al (2013) The circadian clock goes genomic. Genome Biol 14:208

    Article  PubMed Central  PubMed  Google Scholar 

  13. Hsu PY, Harmer SL (2014) Wheels within wheels: the plant circadian system. Trends Plant Sci 19(4):240–9

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  14. James AB, Syed NH, Bordage S et al (2012) Alternative splicing mediates responses of the Arabidopsis circadian clock to temperature changes. Plant Cell 24:961–981

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  15. James A, Syed N, Brown J et al (2012) Thermoplasticity in the plant circadian clock: How plants tell the time-perature. Plant Signal Behav 7:1219–1223

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  16. Sanchez SE, Petrillo E, Beckwith EJ et al (2010) A methyl transferase links the circadian clock to the regulation of alternative splicing. Nature 468:112–116

    Article  CAS  PubMed  Google Scholar 

  17. Jones MA, Williams BA, McNicol J et al (2012) Mutation of Arabidopsis SPLICEOSOMAL TIMEKEEPER LOCUS1 causes circadian clock defects. Plant Cell 24:4907–4916

    Article  PubMed Central  PubMed  Google Scholar 

  18. Staiger D, Heintzen C (1999) The circadian system of Arabidopsis thaliana: forward and reverse genetic approaches. Chronobiol Int 16:1–16

    Article  CAS  PubMed  Google Scholar 

  19. Schmal C, Reimann P, Staiger D (2013) A circadian clock-regulated toggle switch explains AtGRP7 and AtGRP8 oscillations in Arabidopsis thaliana. PLoS Comput Biol 9:e1002986

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  20. Werneke JM, Chatfield JM, Ogren WL (1989) Alternative mRNA splicing generates the two ribulosebisphosphate carboxylase/oxygenase activase peptides in spinach and Arabidopsis. Plant Cell 1:815–825

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  22. Kim SH, Koroleva OA, Lewandowska D et al (2009) Aberrant mRNA transcripts and the nonsense-mediated decay proteins UPF2 and UPF3 are enriched in the nucleolus. Plant Cell 21:2045–2057

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Work in our laboratories is supported by grants from the Biotechnology and Biological Sciences Research Council (BB/G024979/1, European Research Area network Plant Genomics [Plant Alternative Splicing and Abiotic Stress]) and the Scottish Government Rural and Environment Science and Analytical Services division (to J.W.S.B. and C.S.) and the DFG (STA 653 and SPP1530) (to D.S.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Craig G. Simpson .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Simpson, C.G. et al. (2016). Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes. In: Duque, P. (eds) Environmental Responses in Plants. Methods in Molecular Biology, vol 1398. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3356-3_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3356-3_11

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3354-9

  • Online ISBN: 978-1-4939-3356-3

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics