Fig. 4. Extension of the 3′UTR renders the Rluc reporter mRNA NMD-sensitive in vivo.

a Schematic representation of Rluc reporter constructs with either a 200 nts long (200 + pA) or a 1400 nt-long 3′UTR (1400 + pA), modified for expression in human cells. The first 200 nts of the 3′UTR (green line) are identical in both constructs, the additional 1200 nts (red line) of the 1400 + pA 3′UTR correspond to a head-to-tail duplicated sequence originating from the ampicillin resistance gene. The constructs were cloned into pcDNA3.1(−) expression plasmid, where their expression is controlled by a CMV promoter and a bovine growth hormone polyadenylation signal. b Western blot analysis to monitor UPF1 knockdown efficacy in HeLa cells transiently expressing either 200 + pA or 1400 + pA Rluc reporter mRNA. Cell lysates equivalent to 2 × 10⁵ cells were loaded on a 10% SDS-PAGE, transferred onto a nitrocellulose membrane and probed for UPF1 and beta-actin. c Relative abundance of 200 + pA and 1400 + pA mRNAs in cells depleted for UPF1 (UPF1 KD) normalized to cells with a control knockdown (CTR KD) were measured by RT-qPCR. Mean values ± standard deviations of 3 biological replicates are shown, values of the individual experiments are indicated by dots. (Unpaired, two-sided statistical t-test: 1400 + pA vs 200 + pA p value: 0.0039). Source data are provided as a Source Data File.