Protein synthesis inhibition stabilizes urokinase-type plasminogen activator mRNA. Studies in vivo and in cell-free decay reactions.
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.
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Friedrich Miescher-Institut, Basel, Switzerland.
Nagamine
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D000818:Animals; D015152:Blotting, Northern; D002116:Calcitonin / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D003513:Cycloheximide / Q000494:pharmacology*; D066298:In Vitro Techniques; D010142:Pactamycin / Q000494:pharmacology*; D011132:Polyribosomes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D012260:Ribonucleases / Q000378:metabolism; D013552:Swine; D013997:Time Factors; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D012333:RNA, Messenger; D010142:Pactamycin; D002116:Calcitonin; D003513:Cycloheximide; D012260:Ribonucleases; D014568:Urokinase-Type Plasminogen Activator