Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1, Edc3 and RNA in mutually exclusive interactions

Nucleic Acids Res. 2013 Sep;41(17):8377-90. doi: 10.1093/nar/gkt600. Epub 2013 Jul 12.

Abstract

Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a nontranslatable pool or in their degradation. Removal of the 5' cap structure is a crucial step that commits deadenylated mRNAs to 5'-to-3' degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking-mass spectrometry approach shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • DEAD-box RNA Helicases / chemistry*
  • DEAD-box RNA Helicases / metabolism
  • HEK293 Cells
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • RNA / metabolism*
  • RNA-Binding Proteins / chemistry*
  • RNA-Binding Proteins / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Alignment

Substances

  • EDC3 protein, S cerevisiae
  • PAT1 protein, S cerevisiae
  • RNA-Binding Proteins
  • Saccharomyces cerevisiae Proteins
  • RNA
  • DHH1 protein, S cerevisiae
  • DEAD-box RNA Helicases

Associated data

  • PDB/4BRU
  • PDB/4BRW