A Saccharomyces cerevisiae model suitable for screening of correctors of stop-codon mutations

Abstract

In 039-thalassemia the CAG (glutamine) codon is mutated to UAG stop codon causing a premature arrest of translation and promoting NMD (Nonsense-Mediated mRNA Decay) a mechanism that destabilizes nonsense containing mRNAs. We have recently demonstrated read-through at the premature stop codon (PTC) induced by Geneticin (G418) in K562 clones carrying integrated lentiviral constructs containing the 039-thalassemia globin gene under a minimal LCR (locus control region). These clones were treated with G418 and the production of -globin was assessed by FACS analysis, demonstrating synthesis of full-length -globin (Salvatori, J of Biotechnology and Applied Biochemistry, 2009). This observation was confirmed in erythroid precursor cells from homozygous 039-thalassemia patients, in spite of the low level of adult hemoglobin associated with the decreased abundance of the 039-globin mRNA subjected to NMD (Salvatori, Am J Haematol., 2009). These data support the need for screening of novel molecules able to induce read-through at PTC and, possibly, modulate NMD activities. In the present study, we have taken advantage of the yeast S. cerevisae as a model to monitor the effects of G418 on read-through in yeast strains into which NMD was either functional [NMD (+)] or abolished [NMD (-)] by deletion of the UPF1 gene. These strains were transformed with a plasmid bearing a dual luciferase reporter system. This read-through reporter system is based on the Renilla and Firefly luciferase open reading frames which are separated by either a sense [NS(-)] or a nonsense codon [NS(+)]. Transformants were grown in absence or in presence of scalar concentrations of G418 and yeast lysates were subjected to a dual-luciferase reporter assay. Read-through levels were calculated as the nonsense Firefly/Renilla activities ratio normalised to the sense Firefly/Renilla activities ratio. Increased read-through levels were found when S. cerevisae strains were grown in presence of G418, in agreement with published data on the effects of G418 on the 039-thalassemia nonsense mutation. The proposed yeast model is therefore suitable as a screening system for the identification of novel read-through molecules potentially useful in the therapy of human diseases caused by nonsense mutations