Altus M S, Nagamine Y
Friedrich Miescher-Institut, Basel, Switzerland.
J Biol Chem. 1991 Nov 5;266(31):21190-6.
Inhibition of protein synthesis stabilizes a number of mRNAs, but little is known about the mechanism. To understand the relationship between protein synthesis and mRNA stability, we studied the degradation of calcitonin-induced urokinase-type plasminogen activator (uPA) mRNA in LLC-PK cells. uPA mRNA became highly stable by pretreatment with either cycloheximide or pactamycin, and the stabilizing effect of cycloheximide treatment was time dependent with the full effect exerted by 60 min. Stabilization was also observed with histone H4 mRNA but only partially with c-myc mRNA. To further analyze, we developed a cell-free decay reaction system based on post-mitochondrial supernatant (PMS). In this system, uPA mRNA was completely stable when fractions were obtained from cells pretreated with cycloheximide, but very unstable in control fractions, paralleling uPA mRNA stability in intact cells. However, in contrast to uPA mRNA and the in vivo observation, histone H4 mRNA was unstable whether or not the cells were pretreated with cycloheximide. These results suggest that inhibition of protein synthesis stabilizes mRNAs in at least two different ways in LLC-PK1 cells. When PMS from cycloheximide/calcitonin-treated cells was mixed with PMS from untreated cells, uPA mRNA was not destabilized. This suggests that a putative labile factor responsible for uPA mRNA degradation is not a soluble protein.
蛋白质合成的抑制作用可使多种mRNA稳定，但对其机制却知之甚少。为了解蛋白质合成与mRNA稳定性之间的关系，我们研究了降钙素诱导的尿激酶型纤溶酶原激活剂（uPA）mRNA在LLC-PK细胞中的降解情况。用放线菌酮或春日霉素预处理后，uPA mRNA变得高度稳定，放线菌酮处理的稳定作用具有时间依赖性，60分钟时发挥出完全效应。组蛋白H4 mRNA也观察到了稳定性，但c-myc mRNA仅部分观察到稳定性。为了进一步分析，我们开发了一种基于线粒体后上清液（PMS）的无细胞衰变反应系统。在该系统中，当从用放线菌酮预处理的细胞中获得组分时，uPA mRNA完全稳定，但在对照组分中非常不稳定，这与完整细胞中uPA mRNA的稳定性情况相似。然而，与uPA mRNA及体内观察结果相反，无论细胞是否用放线菌酮预处理，组蛋白H4 mRNA都不稳定。这些结果表明，在LLC-PK1细胞中，蛋白质合成的抑制作用至少以两种不同方式使mRNA稳定。当将放线菌酮/降钙素处理的细胞的PMS与未处理细胞的PMS混合时，uPA mRNA并未变得不稳定。这表明，负责uPA mRNA降解的一种假定不稳定因子不是可溶性蛋白质。
