Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1

Abstract

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

Nonsense-Mediated mRNA Decay: Degradation of Defective Transcripts Is Only Part of the Story

“…The C-terminal PIN domain of Smg6 exhibits nuclease activity in vitro (67), and, in vivo, Smg6’s endonuclease activity cleaves NMD substrates (22, 51, 90, 143, 192). Upf1 phosphorylation is also observed in yeast (47, 123, 218), but these cells lack an ortholog of the Smg1 kinase, and yeast Upf1 also lacks the regions targeted by Smg1. Currently, there is no direct evidence that links Upf1 phosphorylation to NMD regulation in yeast (123, 218).…”

“…Upf1 phosphorylation is also observed in yeast (47, 123, 218), but these cells lack an ortholog of the Smg1 kinase, and yeast Upf1 also lacks the regions targeted by Smg1. Currently, there is no direct evidence that links Upf1 phosphorylation to NMD regulation in yeast (123, 218). …”

Mechanism, factors, and physiological role of nonsense-mediated mRNA decay

“…Even though phosphorylation was also reported for yeast Upf1, the mechanism and the responsible kinase are different. Yeast Upf1 lacks most of the clustered SQ and TQ motifs in the C-terminus and no ortholog of SMG1 has been found [155,156]. The phosphorylation sites in mammalian UPF1 act as recruitment platforms for the remaining core NMD factors, SMG5, SMG6, and SMG7.…”

The evolution and diversity of the nonsense-mediated mRNA decay pathway

“…The lack of S/TQ dipeptides suggest that classical phosphorylation of UPF1 is not required for the activation of NMD in baker’s yeast. Tyrosine phosphorylation of UPF1 in baker’s yeast has been observed and appears to regulate the RNA helicase activity of UPF1 58 . It is possible that these or other phosphorylated sites could act to recruit decay factors like S/TQ dipeptides do.…”