Selective degradation of mRNA: the role of short-lived proteins in differential destabilization of insulin-induced creatine phosphokinase and myosin heavy chain mRNAs during rat skeletal muscle L6 cell differentiation.

This investigation concerns the combined effects of removal and readdition of insulin and inhibition of protein and RNA synthesis on the stability of insulin-induced mRNAs during and after differentiation of rat L6A1 myoblast cells in culture. Addition of insulin accompanying the withdrawal of the mitogenic stimulus of serum to myoblasts caused an 80-fold increase in creatine phosphokinase (CK) activity which was largely accounted for by a similar increase in the amount of CK mRNA. The latter was co-ordinately induced with myosin heavy chain (MHC) mRNA but not malic enzyme (ME) mRNA. Measurements of steady-state levels of mRNA showed that removal of insulin caused CK mRNA, but not MHC mRNA, to be rapidly degraded, the effect being reversed upon readdition of the hormone. Direct measurement of 3H-labeled CK, MHC and beta-actin mRNAs confirmed the selective stabilization and destabilization of CK mRNA by the hormone. Conditions were established for a time-window during which cycloheximide (Cx) produced a virtually total arrest of protein synthesis in myotubes that was reversible upon removal of the inhibitor. Under these conditions, Cx selectively prevented the degradation of CK mRNA in a reversible manner. Actinomycin D (Act D) also arrested the loss of this mRNA. Under the same conditions of mRNA stabilization during de-induction, a superinduction of CK mRNA, but not MHC mRNA, was observed if the two inhibitors were added during induction in the continuous presence of insulin. We conclude that a short-lived protein(s), encoded by a short-lived mRNA(s), selectively regulates the stability of reversibly inducible mRNA.     

Variable stabilities and recoveries of rat-liver RNA polymerases A and B according to growth status of the tissue.

1. The effect of growth status on the relative levels and recoveries of DNA-dependent RNA polymerase in rat liver nuclei was determined by two independent procedures: (a) measurement of RNA polymerase A and B activities in fraction IV [Roeder, R. G. and Rutter, W. J. (1970) Proc. Natl Acad. Sci. U.S.A. 65, 675--682] in the presence and absence of low concentrations of alpha-amanitin; (b) DEAE-Sephadex chromatography of fraction IV to resolve RNA polymerases A and B (and possibly other forms of the enzyme). 2. Growth was arrested in young rats (less than 100 g body weight) by hypophysectomy and stimulated by the administration of growth hormone or triiodothyronine. Under these conditions the rate of RNA synthesis in vivo or in isolated nuclei is known to be markedly depressed or stimulated relatively soon after hypophysectomy or hormone administration, respectively. RNA polymerases were obtained from animals under different growth conditions. There were no differences in the activities of nuclear RNA ploymerases per se, when these were separated from their endogenous template and assayed with heterologous denatured DNA. These reports contrast with earlier reports [Smuckler, E. A. and Tata, J. R. (1971) Nat. New Biol. 234, 37--39; Sajdel, E. M. and Jacob, S. T. (1971) Biochem. Biophys. Res. Commun. 45, 707--715]. 3. The discrepancy was resolved when a 'balance sheet' of enzyme recovery was established. Cessation of growth by hypophysectomy led to a marked reduction in the recovery of both forms A and B of the enzyme (less than 20% of the input RNA polymerase activity in fraction iv) following chromatography on DEAE-Sephadex. This effect was reversed within a short time after the administration of growth hormone (3--9 h) or triiodothyronine (18--24 h), leading to a doubling of the enzyme recoveries. These alterations which were more marked for RNA polymerase A, resulted in different elution profiles for RNA polymerases A and B upon chromatography. 4. It is concluded that the use of DEAE-Sephadex chromatography to compare the levels of RNA polymerases A and B isolated from tissues of different growth rate can give rise to over-estimates of apparent changes in their relative activities and that the measurement of enzyme activity in fraction IV is a better index of RNA polymerase levels. The relationship between growth rate of cells, the stability of RNA polymerases, and the importance of determining enzyme recoveries upon chromatography, are discussed.     

Cell death: Are new proteins synthesized during hormone-induced tadpole tail regression?

The possibility that tadpole tail regression might be initiated by thyroid hormone-induced synthesis of new proteins was investigated. Changes in the newly-synthesized proteins of cultured Xenopus laevis tadpole tails treated with 1.5 × 10^?7 M tri-iodothyronine (T₃) were studied, using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate for protein separation. After initial studies of unfractionated tail proteins, fractionated mixtures of [³H]methionine and [³⁵S]methionine labelled proteins derived from control and hormone-treated tails respectively were examined for hormone-induced changes. Using a new procedure developed to allow effective analysis of small differences in distribution of two isotopes within gel slices, it was shown that no significant changes in synthesis of fractionated tail proteins are induced by the hormone during the first 3–4 days in culture. The average detection limit was approx. 0.02% of total tail protein synthesis. Although no changes in the synthesis of the tissue structural or enzymic proteins are induced by the hormone this study still leaves open the possibility of changes in the synthesis of regulatory proteins. Based on the known method of activation of the tadpole tail collagenase (which is shown here directly for the first time to be involved in T₃-induced tail regression), it is suggested that many of the initial hormone-induced changes might result from T₃-induced activation of proteolytic “cascades”.