Immunopurification and Analysis of Protein and RNA Components of mRNP in Mammalian Cells

  • Fabrice Lejeune
  • Lynne E. Maquat
Part of the Methods in Molecular Biology™ book series (MIMB, volume 257)

Abstract

We describe a basic, fast, and reliable technique to isolate and characterize ribonucleoprotein (RNP) using antibody to a constituent protein. The antibody serves to immunopurify RNP from total cells or nuclear and cytoplasmic cell fractions under conditions that promote RNP integrity. The presence of other RNP proteins as well as transcripts can then be analyzed by Western blotting and reverse transcription polymerase chain reaction, respectively. RNase treatment before immunopurification can be used to assess the dependence of protein-protein interactions on RNA. We also describe a modification using β-mercaptoethanol that facilitates analyzing proteins that comigrate with antibody light or heavy chains.

Key Words

Immunopurification mRNP Western blotting RT-PCR RNase treatment β-mercapto-ethanol 

References

  1. 1.
    Keene, J. D. (2001) Ribonucleoprotein infrastructure regulating the flow of genetic information between the genome and the proteome. Proc. Natl. Acad. Sci. USA 98, 7018–7024.PubMedCrossRefGoogle Scholar
  2. 2.
    Dreyfuss, G., Kim, V. N., and Kataoka, N. (2002) Messenger-RNA-binding proteins and the messages they carry. Nat. Rev. Mol. Cell. Biol. 3, 195–205.PubMedCrossRefGoogle Scholar
  3. 3.
    Ishigaki, Y., Li, X., Serin, TG., and Maquat, L. E. (2001) Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607–617.PubMedCrossRefGoogle Scholar
  4. 4.
    Lejeune, F., Ishigaki, Y., Li, X., and Maquat, L. E. (2002) The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling. EMBO J. 21, 3536–3545.PubMedCrossRefGoogle Scholar
  5. 5.
    Lykke-Andersen, J. (2001) mRNA quality control: Marking the message for life or death. Curr. Biol. 11, R88–R91.PubMedCrossRefGoogle Scholar
  6. 6.
    Maquat, L. E. (2002) Nonsense-mediated mRNA decay. Curr. Biol. 12, R196–R197.PubMedCrossRefGoogle Scholar
  7. 7.
    Fury, M. G. and Zieve, G. W. (1996) U6 snRNA maturation and stability. Exp. Cell Res. 228, 160–163.PubMedCrossRefGoogle Scholar
  8. 8.
    Vallentin, A., Prevostel, C., Fauquier, T., Bonnefont, X., and Joubert, D. (2000) Membrane targeting and cytoplasmic sequestration in the spatiotemporal localization of human protein kinase C alpha. J. Biol. Chem. 275, 6014–6021.PubMedCrossRefGoogle Scholar
  9. 9.
    Sormunen, R. T., Leong, A. S., Vaaraniemi, J. P., Fernando, S. S., and Eskelinen, S. M. (1999) Immunolocalization of the fodrin, E-cadherin, and beta-catenin adhesion complex in infiltrating ductal carcinoma of the breast-comparison with an in vitro model. J. Pathol. 187, 416–423.PubMedCrossRefGoogle Scholar
  10. 10.
    Chu, E., Schmitz, J. C., Ju, J., and Copur, S. M., (1999) An immunoprecipitation-RNA:rPCR method for the in vivo isolation of ribonucleoprotein complexes. Meth. Mol. Biol. 118, 265–274.Google Scholar
  11. 11.
    Serin, G., Gersappe, A., Black, J. D., Aronoff, R., and Maquat, L. E. (2001) Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4). Mol. Cell. Biol. 21, 209–223.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2004

Authors and Affiliations

  • Fabrice Lejeune
    • 1
  • Lynne E. Maquat
    • 1
  1. 1.Department of Biochemistry and Biophysics, School of Medicine and DentistryUniversity of RochesterRochester

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