Role of eucaryotic mRNA cap binding proteins in protein synthesis and regulation in poliovirus infected HeLa cells

The cap structure m('7)GpppX(m)...at the 5' terminus of eucaryotic mRNAs facilitates ribosomes binding to mRNA via interaction with cap binding proteins (CBP). Polypeptides of 24, 50 and 80 kilodaltons in crude initiation factors can be specifically crosslinked to the cap structure of mRNA. Crosslinking of the 50 and 80 kilodalton polypeptides requires ATP hydrolysis, but shows reduced dependence on ATP if the mRNA has less secondary structure. Purification by m('7)GDP affinity chromatography yields the CBP complex, comprising polypeptides of 24, 50 and (TURN)220 kilodaltons. The CBP complex and eucaryotic initiation factor-4B (eIF-4B, 80 kilodaltons) are sufficient to allow a cap specific mRNA protein interaction between the 24 and 50 kilodalton polypeptides of the CBP complex, eIF-4B and mRNA. In relation to the cap specific polypeptides in crude initiation factors, the 50 kilodalton polypeptide is eucaryotic initiation factor-4A (eIF-4A) and the 80 kilodalton polypeptide is most probably eIF-4B. This suggests that the cap binding protein complex and possibly eIF-4B, denature mRNA. In poliovirus-infected cells, uncapped poliovirus RNA is translated when cellular (capped) mRNA translation is inhibited. The 220 kilodalton polypeptide of the CBP complex is proteolyzed in poliovirus-infected cells, correlating with a reduction in the crosslinking of the 24, 50 and 80 kilodalton polypeptides, thus probably explaining the inhibition of cellular mRNA translation. mRNAs with reduced secondary structure are less dependent on the fully active CBP complex for ribosome binding, consistent with the suggestion that the CBP complex can denature capped mRNAs. The data indicate an important role for the 220 kilodalton polypeptide in ribosome binding and, that mRNA secondary structure is a significant determinant in translation of capped mRNAs