Marzena Jankowska

A nuclear cap binding protein complex involved in pre-mRNA splicing

A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.

Fluorescence Studies on Association of Human Translation Initiation Factor eIF4E with mRNA cap-Analogues

Binding of a long series of mono- and dinucleotide analogues of the 7-methylguanosine containing 5′-mRNA-cap to human protein translation initiation factor eIF4E has been investigated by means of fluorescence. A new methodological approach in gathering and analysis of the fluorescence data provided us with very accurate values of the association equilibrium constant K and normalized, maximal quenching of the protein fluorescence ΔFmax, during titration of eIF4E by various cap-analogues. The results confirm participation of at least two conserved tryptophan residues of eIF4E in interaction with 7-methylguanine, as has been described recently for murine eIF4E, complexed with 7-methyl-GDP in crystal (Marcotrigiano et al., 1997, Cell 89, 951), and for yeast eIF4E, complexed with the same ligand in solution (Matsuo et al., 1997, Nature Struct. Biol. 4, 717). On the other hand binding by eIF4E of unmethylated guanine nucleotides and N2,N2,7-trimethylguanine containing nucleotides differ substantially from the way of binding of the regular mRNA-cap. Influence of the structural features of the cap-analogues, especially the type of the second nucleoside in the dinucleotide caps, on their association with eIF4E and biological activities in in vitro protein translation systems has been discussed in light of the known structures of the eIF4E -7- methyl-GDP complexes in crystal and solution.

1H-NMR studies on association of mRNA cap-analogues with tryptophan-containing peptides.

1H-NMR spectroscopy was applied to a study of the mode of interaction, in aqueous medium in the pH range 5.2-8.5 and at low and high temperatures, between several mono- and dinucleotide analogues of the mRNA cap m7GpppG and a selected tripeptide Trp-Leu-Glu, and a tetrapeptide Trp-Glu-Asp-Glu, the sequence of which corresponds to one of the suspected binding sites in the mRNA cap-binding protein (CBP). A program, GEOSHIFT, was developed, based on ring-current anisotropy theory, for analysis of experimentally observed changes in chemical shifts accompanying interactions between aromatic heterocyclic rings. This permitted quantitative evaluation of stacking interactions between the m7G cap and the tryptophan indole ring, and the relative orientations of the planes of the two rings, spaced about 3.2 angstroms apart. The structures of the stacked complexes were determined. In particular, stacking between m(2,2,7)3G (which has no free amino group for hydrogen bonding) and the indole ring is weaker and quite different from that between m7G and m(2,7)2G and indole. With the dinucleotide cap-analogues, only the m7G component stacks with the indole ring, without disruption of intramolecular stacking. In contrast to numerous earlier reports, the calculated stacking interactions are quantitatively in accord with the values derived from fluorescence measurements. It also has been shown that the positively charged (cationic) form of m7G stacks much more efficiently with the indole ring than the zwitterionic form resulting from dissociation of the guanine ring N1H (pKa approximately 7.3).

Studies on Association of mRNA Cap-analogues with a synthetic Dodecapeptide DGIEPMWEDEKN

Abstract 1H NMR and fluorescence were applied to a study of interactions between dinucleotide analogues of mRNA cap and a peptide DGIEPMWEDEKN. Structures of the m7G-Trp complexes were determined by means of the ring current anisotropy theory, and their features were refered to the known X-ray structure of the eIF4E-m7GDP associate.