Biophysical foundations of mRNA - protein interactions in eukaryotes

m7GDP in complex with eIF4E.

People involved
Ryszard Stolarski
Anna Niedzwiecka
Anna Modrak-Wójcik

Collaboration with (in Department of Biophysics)
Edward Darżynkiewicz
Jan Antosiewicz

Further collaboration
Robert E. Rhoads, Department of Biochemistry and Molecular Biology, Louisiana State University Health Center (LSUHSC), Shreveport, Louisiana, U.S.A.;
Richard E. Davis, Department of Biochemistry and Molecular Genetics, University of Colorado, Aurora CO U.S.A.;
Jacek Wójcik, Katarzyna Ruszczyńska, Konrad Zdanowski, Andrzej Ejchart, Nuclear Magnetic Resonance Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Michał Dadlez, Department of Biophysics and Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland
Anna Niedzwiecka, Michał Wojciechowski, Marek Cieplak, Labolatory of Biological Physics (SL4), Institute of Physics, Polish Academy of Sciences, Warsaw, Poland
Joanna Trylska, Maciej Jasinski, CENT I

Keywords
protein factors eIF4E, eIF4G, 4E-BP, CBP
mRNA 5'cap
intermolecular interactions
molecular spectroscopy
analytical ultracentrifugation
thermodynamics of association and microcalorimetry
molecular dynamics and quantum calculations

Research interests

The investigations are focused on molecular mechanisms of specific macromolecular recognition in terms of physical interactions, as well as on thermodynamic stability of protein - RNA complexes in gene expression processes, i. e. mRNA translation, RNA splicing, and intracellular transport. In our studies we use synthetic, chemically modified mRNA 5’ cap analogues (collaboration with prof. E. Darżynkiewicz’s research group) and recombinant protein factors. The achievements in analysis of binary complexes composed of the cap and eukaryotic initiation factor 4E, eIF4E (murine, human. C. elegans, nematode) were based on a new method of fluorescence titration developed by dr. A. Niedźwiecka. This enabled to determine accurate values of the equilibrium association constants and the amounts of the active (nonenzymatic) protein. Determination the contributions to Gibbs free energy of the association from particular stability contacts inside the protein binding centre, and the thermodynamic parameters of association, enthalpy, entropy, heat capacity under constant pressure, led to a two-step binding model. Initial stabilization of the cap via the phosphate chain is subsequently enhanced by cooperative formation of cation - pi and hydrogen bond contacts. Analogous studies on the interactions of cap-binding complex CBC (CBP20 and CBP80 subunits) with selected cap analogues showed a crucial role of the second nucleotide (the first transcribed) as well as the diverse roles of three tyrosines in the recognition and stabilization of the 5’ mRNA terminus by CBP20. Differences in the specific binding of two caps, monomethylguanosine (MMG-cap) and trimethylguanosine (TMG-cap), by highly homologous eIF4E isoforms from nematodes and C. elegans were initially resolved by molecular dynamics simulations. The dual specificity toward both MMG-cap and TMG-cap vs. monospecifcity toward MMG-cap arises form dynamics of the loops at the entrance to eIF4E binding pocket. NMR experiments together with theoretical calculations of chemical shifts, coupling constants, and electronic charge distribution allowed to localized unequivocally on N(7) the additional positive charge in 7-methylguanine moiety, contrary to the previous model of the positive charge diffused between N(7) and N(9). Our present researches are more directed to ternary complexes composed of mRNA 5’ kap, eIF4E, and 4E-BP (4E binding protein) or large multidomain fragments of eIF4GI. In particular, by means of analytical centrifugation, the cooperativity of the two eIF4E binding sites was shown to depend on the sequence of incorporation the cap and 4E-BP1.

Selected publications

  1. Modrak-Wojcik, A., Gorka, M., Niedzwiecka, K., Zdanowski, K., Zuberek, J., Niedzwiecka, A., Stolarski, R.: Eukaryotic translation initiation is controlled by cooperativity effects within ternary complexes of 4E-BP1, eIF4E, and the mRNA 5’ cap, FEBS Letters 587, 3928-3934, 2013.
  2. Liu, W., Jankowska-Anyszka, M., Piecyk, K., Dickson, L., Wallace, A., Niedźwiecka, A., Stępiński, J., Stolarski, R., Darżynkiewicz, E., Kieft, J., R. Zhao, R., Jones, D.N.M., Davis, R.E.: Structural Basis for Nematode eIF4E binding an m2,2,7G-Cap and its implications for translation initiation, Nucleic Acids Research 39, 8820-8832, 2011.
  3. Kiraga-Motoszko, K., Niedzwiecka, A. Modrak-Wojcik, A., Stepinski, J., Darzynkiewicz, E., Stolarski, R.: Thermodynamics of molecular recognition of mRNA 5' cap by yeast eukaryotic initiation factor 4E, Journal of Physical Chemistry B 115, 8746-8754, 2011.
  4. Ruszczynska-Bartnik K, Maciejczyk M, R. Stolarski: Dynamical insight into Caenorhabditis elegans eIF4E recognition specificity for mono- and trimethylated structures of mRNA 5' cap, Journal of Molecular Modeling 17, 727-737, 2011.
  5. Worch R., Jankowska-Anyszka M., Niedzwiecka A., Stepinski J., Mazza C., Darzynkiewicz E., Cusack S., Stolarski R.: Diverse role of three tyrosines in binding of the RNA 5' cap to the human nuclear cap binding complex, Journal of Molecular Biology 385, 618-627, 2009.
  6. Worch, R. Stolarski, R.: Stacking efficiency and flexibility analysis of aromatic amino acids in cap-binding proteins, Proteins: Structure, Function, Bioinformatics 71, 2026-2037, 2008.
  7. Rutkowska-Włodarczyk, I., Stępiński, J., Dadlez, M., Darżynkiewicz, E., Stolarski, R., Niedźwiecka A.: Structural changes of eIF4E upon binding to the mRNA 5'monomethylguanosine and trimethylguanosine cap, Biochemistry 47, 2710-2720, 2008.
  8. Niedzwiecka, A.,Stępiński, J., Antosiewicz, J.M., Darżynkiewicz, E., Stolarski R.: Biophysical approach to studies of cap-eIF4E interaction by synthetic cap analogs, Methods in Enzymology 430, 209-245, 2007.
  9. Worch, R., Niedzwiecka, A., Stepinski, J., Mazza, C., Jankowska-Anyszka, M., Darzynkiewicz, E., Cusack, S. and Stolarski, R.: Specificity of recognition of mRNA 5' cap by human nuclear cap-binding complex. RNA 11, 1355-1363, 2005
  10. Westman, B., Beeren, L., Grudzien, E., Stepinski, J., Worch, R., Zuberek, J., Jemielity, J., Stolarski, R., Darzynkiewicz, E., Rhoads, R.E. and Preiss, T.: The antiviral drug ribavirin does not mimic the 7-methylguanosine moiety of the mRNA cap structure in vitro. RNA 11, 1505-1513, 2005.
  11. Niedzwiecka, A., Darzynkiewicz, E., and Stolarski, R.: Thermodynamics of mRNA 5' Cap Binding by Eukaryotic Translation Initiation Factor eIF4E. Biochemistry 43, 13305-13317, 2004.
  12. Zuberek, J. Jemielity, J., Jablonowska, A., Stepinski, J., Dadlez, M., Stolarski, R., and Darzynkiewicz, E.: Influence of Electric Charge Variation at Residues 209 and 159 on the Interaction of eIF4E with the mRNA 5' Terminus. Biochemistry 43, 5370-5379, 2004
  13. Stolarski R.: Thermodynamics of Specific protein-RNA interactions. Acta Biochimica Polonica 50, 297-318, 2003
  14. Zuberek, J., Wyslouch-Cieszynska, A., Niedzwiecka, A., Dadlez, M., Stepinski, J., Augustyniak, W., Gingras, A.-C., Zhang, Z., Burley, S.K., Sonenberg, N., Stolarski, R., and Darzynkiewicz E. : Phosphorylation of eIF4E Attenuates Its Interaction with mRNA 5' Cap Analogs by Electrostatic Repulsion: Intein-mediated Protein Ligation Strategy to Obtain Phosphorylated Protein. RNA 9, 52-61, 2003
  15. Jemielity, J.,Fowler, T., Zuberek, Stepinski, J., Lewdorowicz, M., Niedzwiecka, A., Stolarski, R., Darzynkiewicz, E., and Rhoads, R.E. : Novel "Anti-Reverse" Cap Analogs with Superior Translational Properties. RNA 9, 1108-1122, 2003
  16. Ruszczynska, K., Kamienska-Trela, K., Wojcik, J. Stepinski, J., Darzynkiewicz, E., and Stolarski, R.: Charge Distribution in 7-Methylguanine Regarding Cation-pi Interaction with Protein Factor eIF4E. Biophysical Journal 85, 1450-1456, 2003
  17. Niedzwiecka, A., Stepinski, J., Darzynkiewicz, E., Sonenberg, N., and Stolarski, R.: Positive heat capacity change upon specific binding of translation initiation factor eIF4E to mRNA 5' cap. Biochemistry 41 (40):12140-12148, 2002.
  18. Niedzwiecka, A., Marcotrigiano, J., Stepinski, J., Jankowska-Anyszka, M., Wyslouch- Cieszynska, A., Dadlez, M., Gingras, A.-C., Mak, P., Darzynkiewicz, E., Sonenberg, N., Burley, S.K. & Stolarski, R.: Biophysical Studies of eIF4E cap-Binding Protein: Recognition of mRNA 5' cap Structure and Synthetic Fragments of eIF4G and 4E-BP1 Proteins, Journal of Molecular Biology 319, 615-635, 2002
  19. Stępiński, J., Waddell, C., Stolarski, R., Darżynkiewicz, E. & Rhoads, R.E.: Synthesis and Properties of mRNAs Containing the Novel "Anti-Reverse" cap Analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'-deoxy)GpppG, RNA 7, 1486-1495, 2001