Identification of Specific Protein-RNA Target Sites Using Libraries of Natural Sequences

  • Lucy G. Andrews
  • Jack D. Keene
Part of the Methods in Molecular Biology™ book series (MIMB, volume 118)

Abstract

RNA-binding proteins are involved in a variety of regulatory and developmental processes such as RNA processing, transport, and translation and are integral components of ribosomes, spliceosomes, nucleoli, and other ribonucle-oprotein particles. Proteins have been shown to interact directly with mRNA at all stages from the nascent transcript through capping, polyadenylation and splicing, nuclear export, translation initiation, and translocation of ribosomes along the message as well as during degradation of the mRNA (for reviews see refs. 1, 2, 3, 4, 5). It is becoming increasingly clear that many RNA-binding proteins regulate gene expression through their interactions with mRNAs at various stages of development. The classic example is that of the iron-response element mRNA-binding protein, aconitase, which was shown by Klausner and co-workers to regulate translation of ferritin mRNA by binding to a stem-loop structure in its 5′ untranslated region (UTR) (reviewed in ref. 6). Additional examples include the regulated stability of transferrin, histone, and various cytokine and proto-oncogene mRNAs that are controlled by the binding of specific proteins to consensus sequences in their 3′ untranslated regions (3′ UTR) (7, 8, 9). In addition, studies of tobacco mosaic virus (10), the Drosophila developmental gene hunchback (11), 15-lipogenase (12), and various cytokine mRNAs (13) have implicated RNA-binding proteins in modulation of translational efficiency by interaction with sequences within the 3′ UTR.

Keywords

Vortex Phenol Urea Tyrosine Chloroform 

References

  1. 1.
    Bernstein, P. L. and Ross, J. (1989) Poly(A), poly(A) binding protein and the regulation of mRNA stability. Trends Biochem. Sci. 14, 373–377.PubMedCrossRefGoogle Scholar
  2. 2.
    Bingham, P. M., Chou, T. B., Mims, I., and Zachar, Z. (1988) On/off regulation of gene expression at the level of splicing. Trends Genet. 4, 134–138.PubMedCrossRefGoogle Scholar
  3. 3.
    Clawson, G A., Feldherr, C. M., and Smuckler, E. A. (1985) Nucleocytoplasmic RNA transport. Mol. Cell. Biochem. 67, 87–99.PubMedCrossRefGoogle Scholar
  4. 4.
    McCarthy, J. E. and Kollmus, H. (1995) Cytoplasmic mRNA-protein interactions. Trends Biochem. Sci. 20, 191–197.PubMedCrossRefGoogle Scholar
  5. 5.
    Frankel, A. D., Mattaj, I. W., and Rio, D. C. (1991) RNA-protein interactions. Cell 67, 1041–1046.PubMedCrossRefGoogle Scholar
  6. 6.
    Melefors, O. and Hentze, M. W., (1993) Translational regulation by mRNA/protein interactions in eukaryotic cells: ferritin and beyond. Bioessays 15, 85–90.PubMedCrossRefGoogle Scholar
  7. 7.
    Casey, J. L., Hentze, M. W., Koeller, D. M., Caughman, S. W., Rouault, T. A., Klausner, R. D., and Harford, J. B. (1988) Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation. Science 240, 924–928.PubMedCrossRefGoogle Scholar
  8. 8.
    Pandey, N. B. and Marzluff, W. F. (1987) The stem-loop structure at the 3′ end of the histone mRNA is necessary and sufficient for regulation of histone mRNA stability. Mol. Cell. Biol. 7, 4557–4559.PubMedGoogle Scholar
  9. 9.
    Shaw, G. and Kamen, R. (1986) A conserved AU sequence from the 3′ untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659–667.PubMedCrossRefGoogle Scholar
  10. 10.
    Gallie, D. R. and Walbot, V. (1990) RNA pseudoknot domain of tobacco mosaic virus functionally substitutes for a poly(A) tail in plant and animal cells. Genes Dev. 4, 1149–1157.PubMedCrossRefGoogle Scholar
  11. 11.
    Murata, Y. and Wharton, R. P. (1995) Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell 80, 747–756.PubMedCrossRefGoogle Scholar
  12. 12.
    Ostareck-Lederer, A., Ostareck, D. H., Standart, N., and Thiele, B. J. (1994) Transcription of 15-lipoxygenase mRNA is inhibited by a protein that binds to a repeated sequence in the 3′ untranslated region. EMBO J. 13, 1476–1481.PubMedGoogle Scholar
  13. 13.
    Kruys, V. M., Wathelet, M. G, and Huez, G A. (1988) Identification of a translation inhibitory element (TIE) in the 3′ untranslated region of the human interferon-β mRNA. Gene 72, 191–200.PubMedCrossRefGoogle Scholar
  14. 14.
    Kenan, D. J., Query, C. C, and Keene, J. D. (1991) RNA recognition: towards identifying determinants of specificity. Trends Biochem. Sci. 16, 214–220.PubMedCrossRefGoogle Scholar
  15. 15.
    Robinow, S., Campos, A. R., Yao, K.-M., and White, K. (1988) The elav gene product of Drosophila, required in neurons, has the RNP consensus motifs. Science 242, 1570–1572.PubMedCrossRefGoogle Scholar
  16. 16.
    Szabo, A., Dalmau, J., Manley, G, Rosenfeld, M., Wong, E., Henson, J., Posner, J. B., and Furneaux, H. M. (1991) HuD, a paraneoplastic encephalomyelitis antigen, contains RNA-binding domains and is homologous to sex-lethal. Cell 67, 325–333.PubMedCrossRefGoogle Scholar
  17. 17.
    Sakai, K., Gofuku, M., Kitagawa, Y., Ogasawara, T., Hirose, G., Yamazaki, M., Koh, C-H., Yanagisawa, N., and Steinman, L. (1994) A hippocampal protein associated with paraneoplastic neurologic syndrome and small lung carcinoma. Biochem. Biophys. Res. Commun. 199, 1200–1208.PubMedCrossRefGoogle Scholar
  18. 18.
    Ma, W.-J., Cheng, S., Campbell, C, Wright, A., and Furneaux, H. (1996). Cloning and characterization of HuR, a ubiquitously expressed Elav-like protein. J. Biol. Chem. 271, 1–8.Google Scholar
  19. 19.
    Tsai, D. E., Harper, D. S., and Keene, J. D. (1991) U1-snRNP-A protein selects a ten nucleotide consensus sequence from a degenerate RNA pool presented in various structural contexts. Nucleic Acids Res. 19, 4931–4936.PubMedCrossRefGoogle Scholar
  20. 20.
    Andrews, L. G. and Keene, J. D. (1997) Interactions of proteins with specific sequences in RNA, in mRNA Formation and Function (Richter, J. D., ed.). Academic Press, San Diego, pp. 237–261.CrossRefGoogle Scholar
  21. 21.
    Levine, T. D., Gao, F-B., King, P. H., Andrews, L. G., and Keene, J. D. (1993) Hel-N1: an autoimmune RNA-binding protein with specificity for 3′ uridylate-rich untranslated regions of growth factor mRNAs. Mol. Cell. Biol. 13, 3494–3504.PubMedGoogle Scholar
  22. 22.
    Keene, J. D. (1996) Randomization and selection of RNA to identify targets for RRM RNA-binding proteins and antibodies. Methods Enzymol. 267, 367–383.PubMedCrossRefGoogle Scholar
  23. 23.
    Gao, F.-B., Carson, C. C., Levine, T. D., and Keene, J. D. (1994) Selection of a subset of mRNAs from combinatorial 3′ untranslated region libraries using neuronal RNA-binding protein, Hel-N1. Proc. Natl. Acad. Sci. USA 91, 11,207–11,211.PubMedCrossRefGoogle Scholar
  24. 24.
    Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  25. 25.
    Wang, Y.-X., Lu, M., and Draper, D. E. (1993) Specific ammonium ion requirement for functional ribosomal RNA tertiary structure. Biochemistry 32, 12,279–12,282.PubMedCrossRefGoogle Scholar
  26. 26.
    Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.PubMedGoogle Scholar
  27. 27.
    Keene, J. D. (1996) RNA surfaces as functional mimetics of proteins. Chem. Biol. 3, 505–513.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 1999

Authors and Affiliations

  • Lucy G. Andrews
    • 1
  • Jack D. Keene
    • 2
  1. 1.Department of BiologyThe University of Alabama at BirminghamBirminghamUSA
  2. 2.Department of Microbiology, Combinatorial Sciences CenterDuke University Medical CenterDurhamUSA

Personalised recommendations