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 pg/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 eEFTu in erythroleukemic cells is controlled, at least in part, by a translational mechanism.

Regulation of the utilization of mRNA for eucaryotic elongation factor Tu in Friend erythroleukemia cells.

“…The sucrose gradient buffer contained 50 mM Tris hydrochloride, pH 7.5, 100 mM NaCl, and 3 mM magnesium acetate. Gradients were centrifuged at 41,000 rpm for 2.5 h at 4°C in an SW41 rotor and fractionated as described previously (39). RNA from fractions 5 through 27 (bottom of gradient) were prepared (0.4 nil per fraction) and used for dot blot analysis as described in Materials and Methods.…”

“…FEL cells were grown at 37°C in Ham F-10 medium containing 5% bovine calf serum as described previously (39). Cell number was determined with a hemacytometer.…”

“…Polyadenylated [poly(A)+] RNA was prepared by two cycles of oligo(dT)-cellulose chromatography. Polysomal and mRNP RNAs were recovered from sucrose gradient fractions of cytoplasmic extracts previously centrifuged on 10 to 40% sucrose gradients (39). To purify the RNA from sucrose gradients for dot blots, RNPs were precipitated by adding 2.5 volumes of 95% ethanol at -20°C.…”

“…Cell number was determined with a hemacytometer. Cytoplasmic extracts were prepared by lysing cells with Triton X-100 in the presence of 6.6 mM vanadyl ribonucleoside, as described elsewhere (39).…”

“…(39). These results, however, were based on translation assays, i.e., on the activity of a single mRNA in the presence of a large amount of other mRNAs, and therefore suffered from the limitations such an assay imposes.…”