Identification of novel phosphorylation sites in the mRNA surveillance factor Upf2 from Saccharomyces cerevisiae

Abstract

Cells are not exempt from committing errors. One of these errors is the addition of a premature termination codon (PTC) to an mRNA. These PTC‐harboring mRNAs encode potential non‐functional proteins that can lead to disease. Cells rely on the nonsense‐mediated mRNA decay (NMD) pathway to prevent the synthesis of these proteins. NMD is executed by three major proteins: Upf1, Upf2 and Upf3. Evidence has shown that Upf2 is phosphorylated in yeast and mammals but the role of this modification is unclear. The goal of this project is to provide insight on the molecular and biochemical basis of Upf2 phosphorylation and its role in NMD activity. Mass spectrometry analysis of immunopurified Upf2 from S. cerevisiae revealed twelve novel phosphorylation sites (S54, S55, T327, S422, T842, S868, T869, T872, S874, S1021, S1022 and S1023). Residues T842, S868, T869, T872 and S874 are located in the Upf2 acidic domain, previously reported to be phosphorylated. In contrast, S1021, S1022 and S1023 locate in the carboxyl terminal, a region important for NMD activity. Mutations of these residues are being constructed by site‐directed PCR‐mediated mutagenesis to alter the phosphorylated residues and assess their role in NMD. These results suggest that Upf2 phosphorylation is conserved in S. cerevisiae and might play an important role in mRNA surveillance. Supported by U54 CA96297, PRLSAMP HRD‐0601843, and RISE 2R25GM61151.