Figure 8: The degradation induced by TNF-α and IL6 3′ UTRs involves endocleavage.
From: Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences
(a,d) TPI reporter mRNAs with xrRNA-framed RAB7A (control), TNF-α or IL6 (decay-inducing) 3′ UTRs are represented as in Fig. 1, with the inserted sequences shown as coloured boxes. Dual xrRNA elements (xrA and xrB) are indicated. (d) Deletion mutants are shown containing the stem–loop, marked as a hairpin, or the cleavage site, marked by a schematic endonuclease. (b,c; e,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. The reporter mRNA expression was induced for 24 h with 1 μg ml−1 doxycycline (Dox). Knockdown was performed by transfecting the indicated siRNA. (c,e) Mean values of reporter and xrFrag signal±s.d. (n=3) were quantified and normalized to RAB7A control. The ratio of xrFrag to reporter mRNA levels is indicated below the graph.
