Identification of novel members of the 5 - TOP family of mRNAs and their regulation by mTORC1 signalling

We have employed a stable isotope- labelling approach to study the impact of rapamycin and mTOR kinase inhibitors (mTOR- Kls) on protein synthesis in Hela cells. As expected, rapamycin impaired the synthesis of proteins encoded by mRNAs containing S\u27- terminal oligopyrimidines (5\u27-TOPs), but, surpri singly, mTOR- Kls inhibited this much more strongly. Synthesis of several other proteins displayed the same pattern and, importantly, their mRNAs appear to contain a 5\u27- TOP, indicating they are novel members of this class of mRNAs. The mRNAs for translationally controlled tumour protein TCTP, an anti - apoptotic protein that is up- regulated by serum stimulation and in cancer, also contain a 5\u27-TOP. The seruminduced increase in TCTP levels is partially inhibited by rapamycin and completely by mTORKls. Polysome profile analysis combined with northern blotting revealed that these effects on TCTP and other novel 5\u27 -TOP mRNA- encoded proteins are exerted at the translational level. Consistent with control by mTORCl, TCTP levels are not down- regulated by serumstarvation in mouse embryo fibroblasts lacking tuberous sclerosis complex 2 (TSC2). Although elF4E overexpression enhances the expression of TCTP and the polysomal association of other 5\u27-TOP mRNAs, it does not \u27rescue \u27 their translation from the effects of mTOR inhibition, indicating that additional regulatory mechanisms account for th eir control by mTOR