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

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.

Human DDX6 effects miRNA-mediated gene silencing via direct binding to CNOT1

“…4D). In stark contrast to the defect in CNOT subunit association with MBP-DDX6-Arg386Glu, the association of the mutant protein with Pat1b and EDC3, factors that directly interact with the DDX6 C-terminal lobe (Tritschler et al 2009;Sharif et al 2013), were not impaired (Fig. 4D,E).…”

“…Dhh1p and metazoan Pat1 proteins have been likely candidates due to their abilities to enhance decapping and associate with CCR4-NOT (Coller et al 2001;Fischer and Weis 2002;Haas et al 2010;Nissan et al 2010;Ozgur et al 2010). Pat1 directly binds to DDX6 (Sharif et al 2013), suggesting that it may associate indirectly with CCR4-NOT via DDX6. Interestingly, Pat1 and the decapping enhancer EDC3 both compete for the same binding site on DDX6 (Sharif et al 2013).…”

“…Pat1 directly binds to DDX6 (Sharif et al 2013), suggesting that it may associate indirectly with CCR4-NOT via DDX6. Interestingly, Pat1 and the decapping enhancer EDC3 both compete for the same binding site on DDX6 (Sharif et al 2013). Based on these observations we posit that, upon completion of miRNA-mediated deadenylation by the CCR4-NOT complex, direct recruitment of DDX6, and perhaps Pat1b, assists in dissociation of the cap-binding complex.…”

Polysomes, Stress Granules, and Processing Bodies: A Dynamic Triumvirate Controlling Cytoplasmic mRNA Fate and Function

“…Simultaneously, PAT1 interacts with the DEAD-box RNA helicase DHH1/ DDX6. Both PAT1 and DDH1 play a role in translational inhibition as well as PB formation (Coller and Parker, 2005;Ozgur et al, 2010;Sharif et al, 2013). This links deadenylation with translational repression and decapping.…”

Numerous interactions act redundantly to assemble a tunable size of P bodies in Saccharomyces cerevisiae

“…To understand how additional components could promote P-body assembly, we focused on determining the interactions that allow Dhh1 to rescue P-body formation. Dhh1 is a member of the DEAD-box family of ATPdependent helicases, and is known to interact with Edc3, Pat1, Not1, and RNA, as well as possessing ATPase activity (10,(21)(22)(23). Dhh1 has been suggested to promote P-body assembly by binding ATP, interacting with RNA, and then forming a higher-order assembly (24).…”