Figure 7: The degradation induced by TNF-α and IL6 3′ UTRs is translation dependent.

(a) TPI reporter mRNAs with RAB7A (control), TNF-α or IL6 (decay-inducing) 3′ UTRs are represented as in Fig. 1, with the inserted sequences shown as coloured boxes. (b) Northern blot analysis of RNA samples derived from HeLa cells transiently transfected with the indicated reporter constructs and LacZ as control. (c–f) HeLa Flp-In T-REx cells expressing the indicated reporter RNA were harvested, total RNA extracted and analysed by northern blotting. 7SL RNA serves as endogenous control RNA. Unless indicated otherwise (c,d), reporter mRNA expression was induced for 24 h with 1 μg ml⁻¹ doxycycline (Dox). Cycloheximide treatment together with doxycycline induction was performed for 8 h with 100 μg ml⁻¹ of cycloheximide. Puromycin (Puro) treatment together with doxycycline induction was performed for 4 h with 20 μg ml⁻¹ of puromycin. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to RAB7A control (+Dox for c; 12 h after Dox for d; with or without cycloheximide for e; with or without Puro for f). The ratio of xrFrag to reporter mRNA levels is indicated below the graph.