Figure 6: Isolated ARE and miRNA decay elements induce deadenylation-dependent mRNA degradation.
From: Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences
(a,e) Schematic representation of TPI reporter mRNAs depicted as in Fig. 1, containing (a) the decay-inducing AREs from c-fos and GM–CSF in the 3′ UTR or (e) WT or mutated miRE for let-7 and miR-21. (b,c, f,g) Hela Flp-In T-REx cells expressing the indicated constructs were harvested and RNA was extracted and analysed by northern blotting. (c,g) Knockdown was performed by transfecting the cells with the indicated siRNAs. Expression of reporter mRNAs was induced with 1 μg ml−1 doxycycline (+Dox) for 24 h. Endogenous 7SL RNA levels are shown as a control. Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to the WT control (+Dox for b and f; per knockdown condition for c and g). The ratio of xrFrag to reporter mRNA levels is indicated below the graph. (d) Semi-quantitative PCR analysis of the CNOT1 and GAPDH (control) expression levels in siRNA treated cells.
