Identification and characterization of phosphorylated regions in the nonsense‐mediated mRNA decay protein Upf2 from S. cerevisiae (748.1)

Abstract

About 30% of all mutations causing human diseases generate mRNAs with premature termination codons (PTCs). These PTCs can be incorporated in the mRNA via nonsense or frameshift mutations, normal programmed rearrangements, or biosynthetic errors. As a result, these PTC‐harboring transcripts encode potentially non‐functional and/or toxic proteins that can lead to disease. To avoid the synthesis of these aberrant proteins, the nonsense‐mediated mRNA decay (NMD) pathway targets the PTC‐containing mRNAs for degradation. The core NMD proteins Upf1, Upf2, and Upf3 are evolutionary conserved from lower to higher eukaryotes. Upf2 is a phosphorylated protein in yeast and mammals but the precise role of this post‐translational modification remains elusive. Using tandem mass spectrometry analyses, twelve novel phosphorylation sites (S54, S55, T327, S424, T842, S868, T869, T872, S874, S1021, S1022 and S1023) were identified in Upf2 protein from S. cerevisiae. Furthermore, functional analysis revealed that a small amino‐terminal motif harboring at least two phosphorylated residues is important for NMD activity. Together, these results suggest that Upf2 phosphorylation is a conserved feature of NMD and provide a foundation for future studies to elucidate the functional relevance of Upf2 phosphorylation in the NMD surveillance pathway. Supported by U54 CA96297, PR‐LSAMP HRD‐0601843, and RISE 2R25GM61151.