Using Klenow-Mediated Extension to Measure Poly(A)-Tail Length and Position in the Transcriptome

  • Man Chun Lee
  • Amrei Jänicke
  • Traude Helene Beilharz
Part of the Methods in Molecular Biology book series (MIMB, volume 1125)


The poly(A)-tail that terminates most mRNA and many noncoding RNA is a convenient “hook” to isolate mRNA. However the length of this tail and its position within the primary RNA transcript can also hold diagnostic value for RNA metabolism. In general, mRNA with a long poly(A)-tail is well translated, whereas a short poly(A)-tail can indicate translational silencing. A short poly(A)-tail is also appended to RNA-decay intermediates via the TRAMP complex. A number of approaches have been developed to measure the length and position of the poly(A)-tail. Here, we describe a simple method to “tag” adenylated RNA using the native function of DNA polymerase I to extend an RNA primer on a DNA template in second-strand DNA synthesis. This function can be harnessed as a means to purify, visualize, and quantitate poly(A)-dynamics of individual RNA and the transcriptome en masse.

Key words

ePAT End labeling Polyadenylation Poly(A)-tail length Klenow polymerase Translational control 



We acknowledge members of the Beilharz laboratory for critical discussions. Monash University start-up funds and the Australian Health and Medical Research Council (APP1042851, APP1042848) supported this work, and an Australian Research Fellowship from the Australian Research Council (DP0878224) supported T.H.B.


  1. 1.
    Baker KE, Coller J, Parker R (2004) The yeast Apq12 protein affects nucleocytoplasmic mRNA transport. RNA 10:1352–1358PubMedCentralPubMedCrossRefGoogle Scholar
  2. 2.
    Hector RE, Nykamp KR, Dheur S et al (2002) Dual requirement for yeast hnRNP Nab2p in mRNA poly(A) tail length control and nuclear export. EMBO J 21:1800–1810PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Izawa S, Kita T, Ikeda K et al (2008) Heat shock and ethanol stress provoke distinctly different responses in 3′-processing and nuclear export of HSP mRNA in Saccharomyces cerevisiae. Biochem J 414:111–119PubMedCrossRefGoogle Scholar
  4. 4.
    Beilharz TH, Preiss T (2007) Widespread use of poly(A) tail length control to accentuate expression of the yeast transcriptome. RNA 13:982–997PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Lackner DH, Beilharz TH, Marguerat S et al (2007) A network of multiple regulatory layers shapes gene expression in fission yeast. Mol Cell 26:145–155PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Weill L, Belloc E, Bava FA et al (2012) Translational control by changes in poly(A) tail length: recycling mRNAs. Nat Struct Mol Biol 19:577–585PubMedCrossRefGoogle Scholar
  7. 7.
    Ortiz-Zapater E, Pineda D, Martinez-Bosch N et al (2012) Key contribution of CPEB4-mediated translational control to cancer progression. Nat Med 18:83–90CrossRefGoogle Scholar
  8. 8.
    D’Ambrogio A, Nagaoka K, Richter JD (2013) Translational control of cell growth and malignancy by the CPEBs. Nat Rev Cancer 13:283–290PubMedCrossRefGoogle Scholar
  9. 9.
    Radford HE, Meijer HA, de Moor CH (2008) Translational control by cytoplasmic polyadenylation in Xenopus oocytes. Biochim Biophys Acta 1779:217–229PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Richter JD, Klann E (2009) Making synaptic plasticity and memory last: mechanisms of translational regulation. Genes Dev 23:1–11PubMedCrossRefGoogle Scholar
  11. 11.
    Salles FJ, Richards WG, Strickland S (1999) Assaying the polyadenylation state of mRNAs. Methods 17:38–45PubMedCrossRefGoogle Scholar
  12. 12.
    Beilharz TH, Preiss T (2009) Transcriptome-wide measurement of mRNA polyadenylation state. Methods 48:294–300PubMedCrossRefGoogle Scholar
  13. 13.
    Garneau NL, Sokoloski KJ, Opyrchal M et al (2008) The 3′ untranslated region of sindbis virus represses deadenylation of viral transcripts in mosquito and Mammalian cells. J Virol 82:880–892PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Couttet P, Fromont-Racine M, Steel D et al (1997) Messenger RNA deadenylylation precedes decapping in mammalian cells. Proc Natl Acad Sci U S A 94:5628–5633PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Charlesworth A, Cox LL, MacNicol AM (2004) Cytoplasmic polyadenylation element (CPE)- and CPE-binding protein (CPEB)-independent mechanisms regulate early class maternal mRNA translational activation in Xenopus oocytes. J Biol Chem 279:17650–17659PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Okazaki T, Okazaki R (1969) Mechanism of DNA chain growth. IV. Direction of synthesis of T4 short DNA chains as revealed by exonucleolytic degradation. Proc Natl Acad Sci U S A 64:1242–1248PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Janicke A, Vancuylenberg J, Boag PR et al (2012) ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3′ RACE applications. RNA 18:1289–1295PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Brody JR, Kern SE (2004) Sodium boric acid: a Tris-free, cooler conductive medium for DNA electrophoresis. Biotechniques 36:214–216PubMedGoogle Scholar
  19. 19.
    Sengupta MS, Low WY, Patterson JR et al (2012) ifet-1 is a broad scale translational repressor required for normal P granule formation in C. elegans. J Cell Sci 126:850–859PubMedCrossRefGoogle Scholar
  20. 20.
    Minvielle-Sebastia L, Winsor B, Bonneaud N et al (1991) Mutations in the yeast RNA14 and RNA15 genes result in an abnormal mRNA decay rate; sequence analysis reveals an RNA-binding domain in the RNA15 protein. Mol Cell Biol 11:3075–3087PubMedCentralPubMedGoogle Scholar
  21. 21.
    Yoon OK, Brem RB (2010) Noncanonical transcript forms in yeast and their regulation during environmental stress. RNA 16: 1256–1267PubMedCentralPubMedCrossRefGoogle Scholar
  22. 22.
    Shepard PJ, Choi EA, Lu J et al (2011) Complex and dynamic landscape of RNA polyadenylation revealed by PAS-Seq. RNA 17:761–772PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Beck AH, Weng Z, Witten DM et al (2010) 3′-end sequencing for expression quantification (3SEQ) from archival tumor samples. PLoS One 5:e8768PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Derti A, Garrett-Engele P, Macisaac KD et al (2012) A quantitative atlas of polyadenylation in five mammals. Genome Res 22:1173–1183PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Mangone M, Manoharan AP, Thierry-Mieg D et al (2010) The landscape of C. elegans 3′UTRs. Science 329:432–435PubMedCentralPubMedCrossRefGoogle Scholar
  26. 26.
    Ulitsky I, Shkumatava A, Jan CH et al (2012) Extensive alternative polyadenylation during zebrafish development. Genome Res 22:2054–2066PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Jan CH, Friedman RC, Ruby JG et al (2011) Formation, regulation and evolution of Caenorhabditis elegans 3′UTRs. Nature 469: 97–101PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Tucker M, Valencia-Sanchez MA, Staples RR et al (2001) The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104:377–386PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Man Chun Lee
    • 1
  • Amrei Jänicke
    • 1
  • Traude Helene Beilharz
    • 1
  1. 1.Department of Biochemistry and Molecular BiologyMonash University AustraliaMelbourneAustralia

Personalised recommendations