Figure 5: Characterization of mRNA decay induced by direct protein tethering via xrFrag analysis.
From: Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences
(a,e) TPI and NanoLuc tethering reporter mRNAs with 4MS2 binding sites are depicted as in Fig. 1. (e) The position of the 5′ stem–loop inhibiting ribosome scanning is indicated. (b–d, g,h) Northern blots of RNA samples extracted from HeLa cells transfected with the indicated tethering and reporter constructs. Mean values±s.d. (n=3) for reporter and xrFrag levels were quantified and normalized to tethered GST, which served as control. The ratio of xrFrag to reporter mRNA levels is indicated below the graph. Western blots show the expression levels of the MS2V5-tagged constructs with GFP serving as transfection control. Unspecific bands are indicated with asterisks. (f) Translational efficiency (mean±s.d., n=3) was measured by dual luciferase assay and compared for NanoLuc reporter with or without the 5′ stem–loop. Expression of the NanoLuc mRNAs is shown by northern blotting, co-transfected LacZ served as control.
