Polyadenylation State Microarray (PASTA) Analysis

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 759)

Abstract

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

Key words

Poly(A) tail polyadenylation deadenylation post-transcriptional regulation translational control mRNA stability polysome microarray transcriptome proteome 

References

  1. 1.
    Gebauer, F., and Hentze, M. W. (2004) Molecular mechanisms of translational control. Nat. Rev. Mol. Cell. Biol. 5, 827–835.PubMedCrossRefGoogle Scholar
  2. 2.
    Beach, D. L., and Keene, J. D. (2008) Ribotrap: targeted purification of RNA-specific RNPs from cell lysates through immunoaffinity precipitation to identify regulatory proteins and RNAs. Methods Mol. Biol. 419, 69–91.PubMedCrossRefGoogle Scholar
  3. 3.
    Keene, J. D. (2007) RNA regulons: coordination of post-transcriptional events. Nat. Rev. Genet. 8, 533–543.PubMedCrossRefGoogle Scholar
  4. 4.
    Mathews, M. B., Sonenberg, N., and Hershey, J. W. (2007) Origins and principles of translational control. In: Mathews, M. B., Sonenberg N., and Hershey J. B. W. (eds.), Translational Control in Biology and Medicine (pp. 1–40). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  5. 5.
    Gallie, D. R. (1991) The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency. Genes Dev. 5, 2108–2116.PubMedCrossRefGoogle Scholar
  6. 6.
    Tarun, S. Z., Jr., and Sachs, A. B. (1995) A common function for mRNA 5' and 3' ends in translation initiation in yeast. Genes Dev. 9, 2997–3007.PubMedCrossRefGoogle Scholar
  7. 7.
    Preiss, T., and Hentze, M. W. (1998) Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast. Nature 392, 516–520.PubMedCrossRefGoogle Scholar
  8. 8.
    Gebauer, F., Corona, D. F., Preiss, T., Becker, P. B., and Hentze, M. W. (1999) Translational control of dosage compensation in Drosophila by Sex- lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail. EMBO J. 18, 6146–6154.PubMedCrossRefGoogle Scholar
  9. 9.
    Bergamini, G., Preiss, T., and Hentze, M. W. (2000) Picornavirus IRESes and the poly(A) tail jointly promote cap- independent translation in a mammalian cell-free system. RNA 6, 1781–1790.PubMedCrossRefGoogle Scholar
  10. 10.
    Jacobson, A., and Favreau, M. (1983) Possible involvement of poly(A) in protein synthesis. Nucleic Acids Res. 11, 6353–6368.PubMedCrossRefGoogle Scholar
  11. 11.
    Amrani, N., Ghosh, S., Mangus, D. A., and Jacobson, A. (2008) Translation factors promote the formation of two states of the closed-loop mRNP. Nature 453, 1276–1280.PubMedCrossRefGoogle Scholar
  12. 12.
    Tarun, S. Z., Jr., and Sachs, A. B. (1996) Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G. EMBO J. 15, 7168–7177.PubMedGoogle Scholar
  13. 13.
    Imataka, H., Gradi, A., and Sonenberg, N. (1998) A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A)-binding protein and functions in poly(A)-dependent translation. EMBO J. 17, 7480–7489.PubMedCrossRefGoogle Scholar
  14. 14.
    Richter, J. D., and Sonenberg, N. (2005) Regulation of cap-dependent translation by eIF4E inhibitory proteins. Nature 433, 477–480.PubMedCrossRefGoogle Scholar
  15. 15.
    Hentze, M. W., Gebauer, F., and Preiss, T. (2007) Cis-regulatory sequences and trans-acting factors in translational control. In: Mathews M. B., Sonenberg N., and Hershey J. W. B. (eds.), Translational Control in Biology and Medicine (pp. 269–295). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
  16. 16.
    Goldstrohm, A. C., and Wickens, M. (2008) Multifunctional deadenylase complexes diversify mRNA control. Nat. Rev. Mol. Cell. Biol. 9, 337–344.PubMedCrossRefGoogle Scholar
  17. 17.
    Humphreys, D. T., Westman, B. J., Martin, D. I., and Preiss, T. (2005) MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function. Proc. Natl. Acad. Sci. USA 102, 16961–16966.PubMedCrossRefGoogle Scholar
  18. 18.
    Standart, N., and Jackson, R. J. (2007) MicroRNAs repress translation of m7Gppp-capped target mRNAs in vitro by inhibiting initiation and promoting deadenylation. Genes Dev. 21, 1975–1982.PubMedCrossRefGoogle Scholar
  19. 19.
    Beilharz, T. H., Humphreys, D. T., and Preiss, T. (2009) miRNA effects on mRNA closed-loop formation during translation initiation. In: Rhoads R. E. (ed.), miRNA Regulation of the Translational Machinery (pp. 99–112). Berlin: Springer.Google Scholar
  20. 20.
    Eulalio, A., Huntzinger, E., Nishihara, T., Rehwinkel, J., Fauser, M., and Izaurralde, E. (2009) Deadenylation is a widespread effect of miRNA regulation. RNA 15, 21–32.PubMedCrossRefGoogle Scholar
  21. 21.
    Beilharz, T. H., and Preiss, T. (2009) Transcriptome-wide measurement of mRNA polyadenylation state. Methods 48, 294–300.PubMedCrossRefGoogle Scholar
  22. 22.
    Beilharz, T. H., and Preiss, T. (2007) Widespread use of poly(A) tail length control to accentuate expression of the yeast transcriptome. RNA 13, 982–997.PubMedCrossRefGoogle Scholar
  23. 23.
    Lackner, D. H., Beilharz, T. H., Marguerat, S., et al. (2007) A network of multiple regulatory layers shapes gene expression in fission yeast. Mol. Cell 26, 145–155.PubMedCrossRefGoogle Scholar
  24. 24.
    Beilharz, T. H., and Preiss, T. (2004) Translational profiling: the genome-wide measure of the nascent proteome. Brief Funct. Genomic Proteomic 3, 103–111.PubMedCrossRefGoogle Scholar
  25. 25.
    Mata, J., Marguerat, S., and Bahler, J. (2005) Post-transcriptional control of gene expression: a genome-wide perspective. Trends Biochem. Sci. 30, 506–514.PubMedCrossRefGoogle Scholar
  26. 26.
    Thermann, R., and Hentze, M. W. (2007) Drosophila miR2 induces pseudo-polysomes and inhibits translation initiation. Nature 447, 875–878.PubMedCrossRefGoogle Scholar
  27. 27.
    Binder, R., Horowitz, J. A., Basilion, J. P., Koeller, D. M., Klausner, R. D., and Harford, J. B. (1994) Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3' UTR and does not involve poly(A) tail shortening. EMBO J. 13, 1969–1980.PubMedGoogle Scholar
  28. 28.
    Palatnik, C. M., Storti, R. V., and Jacobson, A. (1979) Fractionation and functional analysis of newly synthesized and decaying messenger RNAs from vegetative cells of Dictyostelium discoideum. J. Mol. Biol. 128, 371–395.PubMedCrossRefGoogle Scholar
  29. 29.
    Minvielle-Sebastia, L., Winsor, B., Bonneaud, N., and Lacroute, F. (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, 3075–3087.PubMedGoogle Scholar
  30. 30.
    Steiger, M. A., and Parker, R. (2002) Analyzing mRNA decay in Saccharomyces cerevisiae. Methods Enzymol. 351, 648–660.PubMedCrossRefGoogle Scholar
  31. 31.
    Sallés, F. J., and Strickland, S. (1995) Rapid and sensitive analysis of mRNA polyadenylation states by PCR. PCR Methods Appl. 4, 317–321.PubMedGoogle Scholar
  32. 32.
    Clancy, J. L., Nousch, M., Humphreys, D. T., Westman, B. J., Beilharz, T. H., and Preiss, T. (2007) Methods to analyze microRNA-mediated control of mRNA translation. Methods Enzymol. 431, 83–111.PubMedCrossRefGoogle Scholar
  33. 33.
    Woolstencroft, R. N., Beilharz, T. H., Cook, M. A., Preiss, T., Durocher, D., and Tyers, M. (2006) Ccr4 contributes to tolerance of replication stress through control of CRT1 mRNA poly(A) tail length. J. Cell. Sci. 119, 5178–5192.PubMedCrossRefGoogle Scholar
  34. 34.
    Tucker, M., Valencia-Sanchez, M. A., Staples, R. R., Chen, J., Denis, C. L., and Parker, R. (2001) The transcription factor associated Ccr4 and Caf1 proteins are components of the major cytoplasmic mRNA deadenylase in Saccharomyces cerevisiae. Cell 104, 377–386.PubMedCrossRefGoogle Scholar
  35. 35.
    Tucker, M., Staples, R. R., Valencia-Sanchez, M. A., Muhlrad, D., and Parker, R. (2002) Ccr4p is the catalytic subunit of a Ccr4p/Pop2p/Notp mRNA deadenylase complex in Saccharomyces cerevisiae. EMBO J. 21, 1427–1436.PubMedCrossRefGoogle Scholar
  36. 36.
    Du, L., and Richter, J. D. (2005) Activity-dependent polyadenylation in neurons. RNA 11, 1340–1347.PubMedCrossRefGoogle Scholar
  37. 37.
    Graindorge, A., Thuret, R., Pollet, N., Osborne, H. B., and Audic, Y. (2006) Identification of post-transcriptionally regulated Xenopus tropicalis maternal mRNAs by microarray. Nucleic Acids Res. 34, 986–995.PubMedCrossRefGoogle Scholar
  38. 38.
    Meijer, H. A., Bushell, M., Hill, K., et al. (2007) A novel method for poly(A) fractionation reveals a large population of mRNAs with a short poly(A) tail in mammalian cells. Nucleic Acids Res. 35, e132.PubMedCrossRefGoogle Scholar
  39. 39.
    Salles, F. J., Richards, W. G., and Strickland, S. (1999) Assaying the polyadenylation state of mRNAs. Methods 17, 38–45.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press 2011

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyMonash UniversityClaytonAustralia
  2. 2.Genome Biology DepartmentThe John Curtin School of Medical Research, The Australian National UniversityActon (Canberra)Australia

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