Environmental Responses in Plants pp 119-132

Part of the Methods in Molecular Biology book series (MIMB, volume 1398) | Cite as

Monitoring Alternative Splicing Changes in Arabidopsis Circadian Clock Genes

  • Craig G. Simpson
  • John Fuller
  • Cristiane P. G. Calixto
  • Jim McNicol
  • Clare Booth
  • John W. S. Brown
  • Dorothee Staiger
Protocol

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.

Key words

Alternative splicing Circadian rhythm RNA-binding proteins Splicing factors 

References

  1. 1.
    Reddy ASN, Marquez Y, Kalyna M et al (2013) Complexity of the alternative splicing landscape in plants. Plant Cell 25:3657–3683PubMedCentralCrossRefPubMedGoogle Scholar
  2. 2.
    Staiger D, Brown JWS (2013) Alternative splicing at the intersection of biological timing, development, and stress responses. Plant Cell 25:3640–3656PubMedCentralCrossRefPubMedGoogle Scholar
  3. 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 10:221PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Hennig L, Menges M, Murray JA et al (2003) Arabidopsis transcript profiling on Affymetrix GeneChip arrays. Plant Mol Biol 53:457–465CrossRefPubMedGoogle Scholar
  5. 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. 6.
    Simpson CG, Fuller J, Maronova M et al (2008) Monitoring changes in alternative precursor messenger RNA splicing in multiple gene transcripts. Plant J 53:1035–1048CrossRefPubMedGoogle Scholar
  7. 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 38:265–278PubMedCentralCrossRefPubMedGoogle Scholar
  8. 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 40:11240–11255PubMedCentralCrossRefPubMedGoogle Scholar
  9. 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:e24638PubMedCentralCrossRefPubMedGoogle Scholar
  10. 10.
    Filichkin SA, Priest HD, Givan SA et al (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20:45–58PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Marquez Y, Brown JWS, Simpson CG et al (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22:1184–1195PubMedCentralCrossRefPubMedGoogle Scholar
  12. 12.
    Staiger D, Shin J, Johansson M et al (2013) The circadian clock goes genomic. Genome Biol 14:208PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Hsu PY, Harmer SL (2014) Wheels within wheels: the plant circadian system. Trends Plant Sci 19(4):240–9PubMedCentralCrossRefPubMedGoogle Scholar
  14. 14.
    James AB, Syed NH, Bordage S et al (2012) Alternative splicing mediates responses of the Arabidopsis circadian clock to temperature changes. Plant Cell 24:961–981PubMedCentralCrossRefPubMedGoogle Scholar
  15. 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 7:1219–1223PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Sanchez SE, Petrillo E, Beckwith EJ et al (2010) A methyl transferase links the circadian clock to the regulation of alternative splicing. Nature 468:112–116CrossRefPubMedGoogle Scholar
  17. 17.
    Jones MA, Williams BA, McNicol J et al (2012) Mutation of Arabidopsis SPLICEOSOMAL TIMEKEEPER LOCUS1 causes circadian clock defects. Plant Cell 24:4907–4916PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Staiger D, Heintzen C (1999) The circadian system of Arabidopsis thaliana: forward and reverse genetic approaches. Chronobiol Int 16:1–16CrossRefPubMedGoogle Scholar
  19. 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:e1002986PubMedCentralCrossRefPubMedGoogle Scholar
  20. 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 1:815–825PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159CrossRefPubMedGoogle Scholar
  22. 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 21:2045–2057PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Craig G. Simpson
    • 1
  • John Fuller
    • 1
  • Cristiane P. G. Calixto
    • 2
  • Jim McNicol
    • 3
  • Clare Booth
    • 1
  • John W. S. Brown
    • 1
    • 2
  • Dorothee Staiger
    • 4
  1. 1.Cell and Molecular SciencesThe James Hutton InstituteInvergowrieUK
  2. 2.Division of Plant SciencesUniversity of Dundee at The James Hutton InstituteDundeeUK
  3. 3.Biomathematics and Statistics ScotlandInvergowrieUK
  4. 4.Molecular Cell Physiology, Faculty of BiologyBielefeld UniversityBielefeldGermany

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