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PAT-Seq: A Method for Simultaneous Quantitation of Gene Expression, Poly(A)-Site Selection and Poly(A)-Length Distribution in Yeast Transcriptomes

  • Angavai Swaminathan
  • Paul F. Harrison
  • Thomas Preiss
  • Traude H. BeilharzEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2049)

Abstract

Next-generation sequencing (NGS) and its application to RNA (RNA-seq) has opened up multiple aspects of RNA processing to deep transcriptome-wide analysis at nucleotide resolution. This has been useful in delineating the transcribed areas of the genome, and in quantitation of RNA isoforms. Such isoforms can diversify the regulatory repertoire of mRNAs. For example, the 3′-end of mRNA can vary in two important ways, in the position chosen for cleavage and polyadenylation, and in the length of the poly(A)-tail. Accordingly, the step-up in resolution made possible by NGS has revealed an unexpectedly high level of alternative polyadenylation (APA). Moreover, it has massively simplified the transcriptome-wide detection of poly(A)-tail length changes. Here we present our approach to the study of 3′-end dynamics using a 3′-focused RNA-seq method called PAT-seq (for poly(A)-test sequencing). The approach records gene expression, APA, and poly(A)-tail changes between transcriptomes to reveal complex interplay between transcriptional and posttranscriptional control mechanisms.

Key words

RNA-seq Alternative polyadenylation Poly(A)-tail Digital gene expression ePAT PAT assay 

Notes

Acknowledgments

We thank members of the Beilharz laboratory for critical feedback. T.P. acknowledges support from the Australian Research Council (ARC Discovery Project DP180100111). T.H.B. is supported by a Biomedicine Discovery Fellowship from Monash University, and acknowledges support the Australian Research Council (ARC Discovery Project DP170100569 and an ARC Future Fellowship FT180100049).

References

  1. 1.
    Harrison PF, Powell DR, Clancy JL, Preiss T, Boag PR, Traven A, Seemann T, Beilharz TH (2015) PAT-seq: a method to study the integration of 3′-UTR dynamics with gene expression in the eukaryotic transcriptome. RNA 21(8):1502–1510.  https://doi.org/10.1261/rna.048355.114CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Beilharz TH, Preiss T (2011) Polyadenylation state microarray (PASTA) analysis. Methods Mol Biol 759:133–148.  https://doi.org/10.1007/978-1-61779-173-4_9CrossRefPubMedGoogle Scholar
  3. 3.
    Beilharz TH, Preiss T (2007) Widespread use of poly(A) tail length control to accentuate expression of the yeast transcriptome. RNA 13(7):982–997.  https://doi.org/10.1261/rna.569407CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Lackner DH, Beilharz TH, Marguerat S, Mata J, Watt S, Schubert F, Preiss T, Bahler J (2007) A network of multiple regulatory layers shapes gene expression in fission yeast. Mol Cell 26(1):145–155. pii: S1097-2765(07)00147-5.  https://doi.org/10.1016/j.molcel.2007.03.002CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Subtelny AO, Eichhorn SW, Chen GR, Sive H, Bartel DP (2014) Poly(A)-tail profiling reveals an embryonic switch in translational control. Nature.  https://doi.org/10.1038/nature13007
  6. 6.
    Chang H, Lim J, Ha M, Kim VN (2014) TAIL-seq: genome-wide determination of poly(A) tail length and 3′ end modifications. Mol Cell 53(6):1044–1052.  https://doi.org/10.1016/j.molcel.2014.02.007CrossRefPubMedGoogle Scholar
  7. 7.
    Gupta I, Clauder-Munster S, Klaus B, Jarvelin AI, Aiyar RS, Benes V, Wilkening S, Huber W, Pelechano V, Steinmetz LM (2014) Alternative polyadenylation diversifies post-transcriptional regulation by selective RNA-protein interactions. Mol Syst Biol 10:719.  https://doi.org/10.1002/msb.135068CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Verma-Gaur J, Qu Y, Harrison PF, Lo TL, Quenault T, Dagley MJ, Bellousoff M, Powell DR, Beilharz TH, Traven A (2015) Integration of posttranscriptional gene networks into metabolic adaptation and biofilm maturation in Candida albicans. PLoS Genet 11(10):e1005590.  https://doi.org/10.1371/journal.pgen.1005590CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dufourt J, Bontonou G, Chartier A, Jahan C, Meunier AC, Pierson S, Harrison PF, Papin C, Beilharz TH, Simonelig M (2017) piRNAs and Aubergine cooperate with Wispy poly(A) polymerase to stabilize mRNAs in the germ plasm. Nat Commun 8(1):1305.  https://doi.org/10.1038/s41467-017-01431-5CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Beilharz TH, Preiss T (2009) Transcriptome-wide measurement of mRNA polyadenylation state. Methods 48(3):294–300. pii: S1046-2023(09)00025-5.  https://doi.org/10.1016/j.ymeth.2009.02.003CrossRefPubMedGoogle Scholar
  11. 11.
    Lee MC, Janicke A, Beilharz TH (2014) Using Klenow-mediated extension to measure poly(A)-tail length and position in the transcriptome. Methods Mol Biol 1125:25–42.  https://doi.org/10.1007/978-1-62703-971-0_3CrossRefPubMedGoogle Scholar
  12. 12.
    Janicke A, Vancuylenberg J, Boag PR, Traven A, Beilharz TH (2012) ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications. RNA 18(6):1289–1295.  https://doi.org/10.1261/rna.031898.111CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Angavai Swaminathan
    • 1
  • Paul F. Harrison
    • 2
  • Thomas Preiss
    • 3
    • 4
  • Traude H. Beilharz
    • 1
    Email author
  1. 1.Development and Stem Cells Program, Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery InstituteMonash UniversityMelbourneAustralia
  2. 2.Monash Bioinformatics PlatformMonash UniversityMelbourneAustralia
  3. 3.Department of Genome Sciences, The John Curtin School of Medical ResearchThe Australian National UniversityCanberraAustralia
  4. 4.Victor Chang Cardiac Research InstituteDarlinghurstAustralia

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