Identification and Functional Analysis of Novel Phosphorylation Sites in the RNA Surveillance Protein Upf1

Nucleic Acids Res. 2014 Feb;42(3):1916-29. doi: 10.1093/nar/gkt1049. Epub 2013 Nov 5.

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Humans
  • Mice
  • Molecular Sequence Data
  • Nonsense Mediated mRNA Decay
  • Peptide Chain Termination, Translational
  • Phosphorylation
  • RNA Helicases / chemistry*
  • RNA Helicases / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Sequence Alignment
  • Tyrosine / metabolism

Substances

  • Saccharomyces cerevisiae Proteins
  • Tyrosine
  • Adenosine Triphosphatases
  • NAM7 protein, S cerevisiae
  • RNA Helicases