Nuclease escape elements protect messenger RNA against cleavage by multiple viral endonucleases
(A) 293T cells were transfected with empty vector (mock) or a plasmid expressing the indicated endonucleases along with a GFP reporter. After 24 h, total RNA was harvested and subjected RT-qPCR to measure GFP mRNA levels. Graphs here and afterwards display individual replicates as dots, together with the mean values (±SEM). Statistical significance was determined by the Student t test (* p<0.1; ** p<0.05; *** p<0.01). (B) 293T cells were co-transfected with the indicated endonuclease-expressing plasmid expressing together with a GFP-SRE or GFP-ΔSRE reporter. After 24 h, total RNA was harvested and subjected RT-qPCR to measure GFP mRNA levels. (C) Top: diagram showing the structure of the reporter mRNA containing MS2 repeats upstream of the SRE or ΔSRE fragment of the IL-6 3’UTR. Red lines denote the region detected by the MS2 probe. Bottom: 293T cells were transfected with the indicated MS2 reporters. 24h later, cells were fixed and processed for RNA FISH staining. Signals from the MS2 probes (red) and DAPI stained nuclei (blue) were detected by confocal microscopy.