They measured Nonsense-mediated mRNA decay efficacy with the one-plasmid screening system, 180ng of a vector carrying the cDNA coding for firefly luciferase and two copies of the same intron located in the coding sequence and in the 3′UTR, are introduced into 8 million U2OS cells with jetOPTIMUS. After 24 h, the cells were distributed into 96-well white clear-bottom plates and treated with cell culture before adding the molecules for an additional 24 h. Then, the luciferase substrate Steadylite Plus was added to each well before reading the luciferase activity with a Tristar luminometer.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades mRNAs carrying a premature termination codon. Its inhibition, alone or in combination with other approaches, could be exploited to develop therapies for genetic diseases caused by a nonsense mutation. This, however, requires molecules capable of inhibiting NMD effectively without inducing toxicity. We have built a new screening system and used it to identify and validate two new molecules that can inhibit NMD at least as effectively as cycloheximide, a reference NMD inhibitor molecule. These new NMD inhibitors show no cellular toxicity at tested concentrations and have a working concentration between 6.2 and 12.5 µM. We have further validated this NMD-inhibiting property in a physiopathological model of lung cancer in which the TP53 gene carries a nonsense mutation. These new molecules may potentially be of interest in the development of therapies for genetic diseases caused by a nonsense mutation.