J M Sierra

Eucaryotic oligonucleotides affecting mRNA translation

Abstract High speed-supernatants and ribosomal salt washes of dormant and developing Artemia salina embryos contain a potent inhibitor of translation; it blocks the elongation factor EF-1-dependent ribosomal binding of aminoacyl-tRNA. A translation activator that counteracts the effect of the inhibitor is found in the same fractions from developing embryos; there is little activator in undeveloped cysts. The appearance of the activator may be responsible for the onset of protein synthesis when development resumes. Both compounds are oligonucleotides. The inhibitor, M r about 6000, is rich in pyrimidines (47% U, 11% A, 26% C, 16% G), sensitive to RNase A, and resistant to RNase T1. The activator, M r about 9000, is rich in guanine (33% U, 10% A, 6% C, 51% G), sensitive to RNase T1, and resistant to RNase A. It complexes with the inhibitor and inactivates it. Inhibitor and activator seem to be end products of hydrolysis of embryo RNA by RNase T1 and RNase A, respectively, and ribosomal salt washes of developing embryos have higher RNase A activity than corresponding fractions from dormant cysts.

Messenger RNA in undeveloped and developing Artemia salina embryos

Summary Messenger activity of RNA species retained [poly (A)+] and not retained [poly (A)−] by oligo(dT) cellulose was assayed in cytoplasmic fractions of Artemia salina embryos using a wheat germ system. In undeveloped embryos, mRNA activity was found in a fraction sedimenting at 15,000 x g (p15) and about half as much in the cytosol (author05). The ribosome (R) fraction, consisting mainly of 80S monomers, had no activity. Only p15 contained [poly (A)+] mRNA amounting to about one tenth of the total mRNA activity of undeveloped embryos. After development for 15–16 hrs, the total mRNA activity of the embryos increased 3-fold. All three cell fractions contained mRNA activity in both [poly (A)−] and [poly (A)+] RNA. Over half of the total was in the polysome-containing R fraction and about one fifth in [poly (A)+] RNA. The most pronounced change with development was in the [poly (A)+] mRNA activity which increased over 6-fold. The patterns of polypeptides synthesized by translation of [poly (A)+] mRNA fractions from undeveloped and developing embryos were similar but not identical.

Translational regulation of the heat shock response

All organisms from bacteria to man respond to an exposure to higher than physiological temperatures by reprogramming their gene expression, leading to the increased synthesis of a unique set of proteins termed heat shock proteins (hsps). The hsps function as molecular chaperones in both normal and stressed cells. The rapid and efficient synthesis of hsps is achieved as a result of changes occurring at gene transcription, RNA processing and degradation, and mRNA translation. With regard to the translational regulation, the emerging picture is that the two key steps of polypeptide chain initiation, namely mRNA binding and Met-tRNAi binding to ribosomes, are regulated in heat-shocked mammalian cells. InDrosophila, mRNA binding is regulated by a structural feature of the leader of heat shock mRNAs and by the inactivation of eukaryotic initiation factor- (eIF-) 4F. No clear evidence for changes in Met-tRNAi binding has been obtained yet.

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts.

Studies on the activation of the heme-stabilized translational inhibitor of reticulocyte lysates by oxidized glutathione and NADPH depletion.

The translational inhibition produced by addition of oxidized glutathione (GSSG) to hemin-containing reticulocyte lysates and the accompanying phosphorylation of the alpha subunit of the polypeptide chain initiation factor eIF-2 can be prevented or reversed by NADPH generators, including glucose 6-phosphate, deoxyglucose 6-phosphate, fructose 6-phosphate, NADPH itself, and also by dithiols, e.g., dithiothreitol, but not by reduced glutathione (GSH) or other monothiols, e.g., 2-mercaptoethanol. The same is true of the inhibition caused by addition of glutamate dehydrogenase, alpha-ketoglutarate, and NH4+, which may be entirely due to NADPH depletion via the reaction.