New reporters for monitoring cellular NMD.

Abstract

Nonsense-mediated decay (NMD) is a eukaryotic surveillance pathway that controls degradation of cytoplasmic transcripts with aberrant features. NMD-controlled RNA degradation acts to regulate a large fraction of the mRNA population. It has been implicated in cellular responses to infections and environmental stress, as well as in deregulation of tumor-promoting genes. NMD is executed by a set of three core factors conserved in evolution, UPF1-3, as well as additional influencing proteins such as kinases. Monitoring NMD activity is challenging due to the difficulties in quantitating RNA decay rates in vivo, and consequently it has also been problematic to identify new factors influencing NMD. Here, we developed a genetic selection system in yeast to capture new components affecting NMD status. The reporter constructs link NMD target sequences with nutrient-selectable genetic markers. By crossing these reporters into a genome-wide library of deletion mutants and quantitating colony growth on selective medium, we robustly detect previously known NMD components in a high-throughput fashion. In addition, we identify novel mutations influencing NMD status and implicate ribosome recycling as important for NMD. By using our constructed combinations of promoters, NMD target sequences, and selectable markers, the system can also efficiently detect mutations with a minor effect, or in special environments. Furthermore, it can be used to explore how NMD acts on targets of different structures.