Translational repression of mRNA for eucaryotic elongation factors in Friend erythroleukemia cells

Abstract

Poly(A)-containing mRNA was prepared from polyribosomes and postpolyribosomal messenger ribonucleoprotein particles (mRNP) from Friend erythroleukemic cells. Both mRNA types were translated in vitro and the 35S-labeled translation products examined by two-dimensional gel electrophoresis. Among the most abundant untranslated mRNA species was the mRNA coding for eucaryotic elongation factor Tu (eEF-Tu). In addition, the mRNA for eucaryotic elongation factor Ts was also present in Friend cells in untranslated form. Calculations based on translation assays indicate that eEF-Tu represents about 15% of the translation products of RNP mRNA and that approximately 40% of the eEF-Tu synthesized in vitro is encoded by translationally repressed mRNA. This repressed mRNA can be activated by addition of cycloheximide to cell cultures. At the level of 0.1 micrograms/ml, cycloheximide was found to inhibit cellular protein synthesis by about 50% while augmenting the relative rate of eEF-Tu synthesis 1.6-fold. This result suggested that eEF-Tu mRNA might initiate poorly. However, addition of supersaturating levels of mRNA to a reticulocyte lysate augmented eEF-Tu synthesis about twofold, while generally depressing the synthesis of other proteins by about 40%. Thus the storage of large amounts of eEF-Tu mRNA in vivo is unlikely to be due directly to the ineffectiveness of the mRNA in competing for the initiation machinery of the cell. The results presented in this report suggest that the supply of active eEF-Tu in erythroleukemic cells is controlled, at least in part, by a translational mechanism.