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Nonradioactive Methods for the Detection of RNA-Protein Interaction

  • Asier Echarri
  • María Eugenia González
  • Iván Ventoso
  • Luis Carrasco
Protocol
  • 141 Downloads
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

RNA in biological systems is associated with proteins. Recent work in eukaryotes has identified common motifs present in families of RNA-binding proteins. Usually, RNA-binding proteins recognize both sequence and structure at their target sites. Therefore, identification of proteins that interact with a specific RNA sequence contributes to the understanding of biological processes. Thus, the genome of human immunodeficiency virus (HIV) encodes proteins, Tat, Rev and NC, that bind to specific viral RNA motives (1,2). These interactions mediate different steps of virus replication i.e., transactivation of transcription, nuclear export of viral transcripts, or packaging of two RNA genomes into the mature virion. In addition, we recently reported that Nef, an accessory protein encoded by HIV-1, belongs to the family of RNA-binding proteins (3). Using different-size variant proteins and point-mutated proteins, it was found that the amino terminal Arg-rich domain of Nef is involved in the RNA-binding activity. Nef proteins from HIV-2 and SIV (simian immunodeficiency virus) also showed RNA-binding capacity (4).

Keywords

Simian Immunodeficiency Virus Nitrocellulose Filter Transfer Buffer Label Riboprobes 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Luban, J. and Goff, S. P. (1991) Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein. J. Virol. 65, 3203–3212.PubMedGoogle Scholar
  2. 2.
    Mattaj, I. W. (1990) A selective review of RNA-protein interactions in eukaryotes. Mol. Biol. Rep. 14, 151–155.PubMedCrossRefGoogle Scholar
  3. 3.
    Echarri, A., González, M. E., and Carrasco, L. (1996) Human immunodeficiency virus (HIV) nef is an RNA binding protein in cell-free systems. J. Mol. Biol. 262, 640–651.PubMedCrossRefGoogle Scholar
  4. 4.
    Echarri, A., González, M. E., and Carrasco, L. (1997) The N-Arg-rich region of human immunodeficiency virus types 1 and 2 and simian immunodeficiency virus Nef is involved in RNA binding. Eur. J. Biochem. 246, 38–44.PubMedCrossRefGoogle Scholar
  5. 5.
    Rodríguez, P. L. and Carrasco, L. (1996) Biotin labeled riboprobes to study RNA-binding proteins, in A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis (Meier, T. and Fahrenholz, F., eds.) Birkháuser Verlag, Basel, Switzerland, 215–225.CrossRefGoogle Scholar
  6. 6.
    Rodríguez, P. L. and Carrasco, L. (1994) Nonradioactive northwestern analysis using biotinylated riboprobes. Biotechniques 17, 702–705.PubMedGoogle Scholar
  7. 7.
    Sambrook, J., Fritsch, E. R., and Maniatis, T., eds. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  8. 8.
    Harlow, E. and Lane, D. (1988) Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  9. 9.
    Lin, J. H., Chang, M. F., Baker, S. C., Govindarajan, S., and Lai, M. M. C. (1990) Characterization of hepatitis delta antigen: Specific binding to hepatitis delta virus RNA. J. Virol. 64, 4051–4058.PubMedGoogle Scholar
  10. 10.
    Southern, E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503–517.PubMedCrossRefGoogle Scholar
  11. 11.
    Chen, X., Sadlock, J., and Schon, E. A. (1993) RNA-binding patterns in total human tissue proteins: Analysis by Northwestern blotting. Biochem. Biophys. Res. Commun. 191, 18–25.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2000

Authors and Affiliations

  • Asier Echarri
    • 1
  • María Eugenia González
    • 2
  • Iván Ventoso
    • 2
  • Luis Carrasco
    • 2
  1. 1.Molecular Biology DepartmentUniversity of MadridMadridSpain
  2. 2.Universidad AutonomaMadridSpain

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