RNA Detection and Visualization pp 221-235

Part of the Methods in Molecular Biology book series (MIMB, volume 714)

In Vivo Visualization of RNA Using the U1A-Based Tagged RNA System

Protocol

Abstract

mRNA transport is a widely used method to achieve the asymmetric distribution of proteins within a cell or organism. In order to understand how RNA is transported, it is essential to utilize a system that can readily detect RNA movement in live cells. The tagged RNA system has recently emerged as a feasible non-invasive solution for such purpose. In this chapter, we describe in detail the U1A-based tagged RNA system. This system coexpresses U1Ap-GFP with the RNA of interest tagged with U1A aptamers, and has been proven to effectively track RNA in vivo. In addition, we provide further applications of the system for ribonucleoprotein complex purification by TAP-tagging the U1Ap-GFP construct.

Key words

U1A GFP Tagged RNA RNA localization ASH1 RNP (ribonucleoprotein) complex 

References

  1. 1.
    Du, T. G., Schmid, M., and Jansen, R. P. (2007) Why cells move messages: the biological functions of mRNA localization. Semin. Cell Dev. Biol. 18, 171–177.PubMedCrossRefGoogle Scholar
  2. 2.
    Kloc, M., Zearfoss, N. R., and Etkin, L. D. (2002) Mechanisms of subcellular mRNA localization. Cell 108, 533–544.PubMedCrossRefGoogle Scholar
  3. 3.
    St Johnston, D. (2005) Moving messages: the intracellular localization of mRNAs. Nat. Rev. Mol. Cell Biol. 6, 363–375.Google Scholar
  4. 4.
    Jambhekar, A., and Derisi, J. L. (2007) Cis-acting determinants of asymmetric, cytoplasmic RNA transport. RNA 13, 625–42.PubMedCrossRefGoogle Scholar
  5. 5.
    Martin, K.C., and Ephrussi, A. (2009) mRNA localization: gene expression in the spatial dimension. Cell 136, 719–730.PubMedCrossRefGoogle Scholar
  6. 6.
    Bullock, S.L. 2007. Translocation of mRNAs by molecular motors: think complex? Semin. Cell Dev. Biol. 18, 194–201.PubMedCrossRefGoogle Scholar
  7. 7.
    Gonsalvez, G. B., Urbinati, C. R., and Long, R. M. (2005) RNA localization in yeast: moving towards a mechanism. Biol. Cell 97, 75–86.PubMedCrossRefGoogle Scholar
  8. 8.
    Chartrand, P., Meng, X. H., Singer, R. H., and Long, R. M. (1999) Structural elements required for the localization of ASH1 mRNA and of a green fluorescent protein reporter particle in vivo. Curr. Biol. 9, 333–336.PubMedCrossRefGoogle Scholar
  9. 9.
    Jansen, R. P., Dowzer, C., Michaelis, C., Galova, M., and Nasmyth, K. (1996) Mother cell-specific HO expression in budding yeast depends on the unconventional myosin myo4p and other cytoplasmic proteins. Cell 84, 687–697.PubMedCrossRefGoogle Scholar
  10. 10.
    Böhl, F., Kruse, C., Frank, A., Ferring, D., and Jansen, R. P. (2000) She2, a novel RNA-binding protein tethers ASH1 mRNA to the Myo4-myosin motor via She3. EMBO J. 19, 5514–5524.PubMedCrossRefGoogle Scholar
  11. 11.
    Long, R.M., Gu, W., Lorimer, E., Singer, R. H., and Chartrand, P. (2000) She2p is a novel RNA-binding protein that recruits the Myo4p-She3p complex to ASH1 mRNA. EMBO J. 19, 6592–6601.PubMedCrossRefGoogle Scholar
  12. 12.
    Takizawa, P. A., and Vale, R. D. (2000) The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p. Proc. Natl. Acad. Sci. USA 97, 5273–5278.PubMedCrossRefGoogle Scholar
  13. 13.
    Boelens, W. C., Jansen, E. J., van Venrooij, W. J., Stripecke, R., Mattaj, I. W., and Gunderson, S. I. (1993) The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA. Cell 72, 881–892.PubMedCrossRefGoogle Scholar
  14. 14.
    Scherly, D., Boelens, W., van Venrooij, W. J., Dathan, N. A., Hamm, J., and Mattaj, I. W. (1989) Identification of the RNA binding segment of human U1A protein and definition of its binding site on U1 snRNA. EMBO J. 8, 4163–4170.PubMedGoogle Scholar
  15. 15.
    Kretzner, L., Krol, A., and Rosbash, M. (1990) Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast specific domains. Proc. Natl. Acad. Sci. USA 87, 851–855.PubMedCrossRefGoogle Scholar
  16. 16.
    Liao, X. C., Tang, J., and Rosbash, M. (1993) An enhancer screen identifies a gene that encodes the yeast U1 snRNP A protein: implications for snRNP protein function in pre-mRNA splicing. Genes Dev. 7, 419–428.PubMedCrossRefGoogle Scholar
  17. 17.
    Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S. M., Singer, R. H., and Long, R. M. (1998) Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445.PubMedCrossRefGoogle Scholar
  18. 18.
    Keryer-Bibens, C., Barreau, C., and Osborne, H. B. (2008) Tethering of proteins to RNAs by bacteriophage proteins. Biol. Cell 100, 125–138.PubMedCrossRefGoogle Scholar
  19. 19.
    Klein Gunnewiek, J. M. T., Hussein, R. I., van Aarssen, Y., Palacios, D., de Jong, R., van Venrooij, W. J., and Gunderson, S. I. (2000) Fourteen residues of the U1 snRNP-specific U1A protein are required for homodimerization, cooperative RNA binding, and inhibition of polyadenylation. Mol. Cell Biol. 20, 2209–2217.PubMedCrossRefGoogle Scholar
  20. 20.
    Coller, J., and Wickens, M. (2007) Tethered function assays: an adaptable approach to study RNA regulatory proteins. Methods Enzymol. 429, 299–321.PubMedCrossRefGoogle Scholar
  21. 21.
    Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Séraphin, B. (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218–229.PubMedCrossRefGoogle Scholar
  22. 22.
    Nagai, K. (1996) RNA-protein complexes. Curr. Opin. Struct. Biol. 6, 53–61.PubMedCrossRefGoogle Scholar
  23. 23.
    Straight, A. F., Sedat, J. W., and Murray, A. W. (1998) Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast. J. Cell Biol. 143, 687–694.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of Cell BiologyYale University School of MedicineNew HavenUSA

Personalised recommendations