A C-terminal silencing domain in GW182 is essential for miRNA function

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182–AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine–tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing

The C-terminal region of Ge-1 presents conserved structural features required for P-body localization

The removal of the 5′ cap structure by the DCP1–DCP2 decapping complex irreversibly commits eukaryotic mRNAs to degradation. In human cells, the interaction between DCP1 and DCP2 is bridged by the Ge-1 protein. Ge-1 contains an N-terminal WD40-repeat domain connected by a low-complexity region to a conserved C-terminal domain. It was reported that the C-terminal domain interacts with DCP2 and mediates Ge-1 oligomerization and P-body localization. To understand the molecular basis for these functions, we determined the three-dimensional crystal structure of the most conserved region of the Drosophila melanogaster Ge-1 C-terminal domain. The region adopts an all α-helical fold related to ARM- and HEAT-repeat proteins. Using structure-based mutants we identified an invariant surface residue affecting P-body localization. The conservation of critical surface and structural residues suggests that the C-terminal region adopts a similar fold with conserved functions in all members of the Ge-1 protein family

Direct role for the Drosophila GIGYF protein in 4EHP-mediated mRNA repression

The eIF4E-homologous protein (4EHP) is a translational repressor that competes with eIF4E for binding to the 5'-cap structure of specific mRNAs, to which it is recruited by protein factors such as the GRB10-interacting GYF (glycine-tyrosine-phenylalanine domain) proteins (GIGYF). Several experimental evidences suggest that GIGYF proteins are not merely facilitating 4EHP recruitment to transcripts but are actually required for the repressor activity of the complex. However, the underlying molecular mechanism is unknown. Here, we investigated the role of the uncharacterized Drosophila melanogaster (Dm) GIGYF protein in post-transcriptional mRNA regulation. We show that, when in complex with 4EHP, Dm GIGYF not only elicits translational repression but also promotes target mRNA decay via the recruitment of additional effector proteins. We identified the RNA helicase Me31B/DDX6, the decapping activator HPat and the CCR4-NOT deadenylase complex as binding partners of GIGYF proteins. Recruitment of Me31B and HPat via discrete binding motifs conserved among metazoan GIGYF proteins is required for downregulation of mRNA expression by the 4EHP-GIGYF complex. Our findings are consistent with a model in which GIGYF proteins additionally recruit decapping and deadenylation complexes to 4EHP-containing RNPs to induce translational repression and degradation of mRNA targets

Molecular basis for GIGYF-Me31B complex assembly in 4EHP-mediated translational repression

GIGYF (Grb10-interacting GYF [glycine-tyrosine-phenylalanine domain]) proteins coordinate with 4EHP (eIF4E [eukaryotic initiation factor 4E] homologous protein), the DEAD (Asp-Glu-Ala-Asp)-box helicase Me31B/DDX6, and mRNA-binding proteins to elicit transcript-specific repression. However, the underlying molecular mechanism remains unclear. Here, we report that GIGYF contains a motif necessary and sufficient for direct interaction with Me31B/DDX6. A 2.4 Å crystal structure of the GIGYF-Me31B complex reveals that this motif arranges into a coil connected to a β hairpin on binding to conserved hydrophobic patches on the Me31B RecA2 domain. Structure-guided mutants indicate that 4EHP-GIGYF-DDX6 complex assembly is required for tristetraprolin-mediated down-regulation of an AU-rich mRNA, thus revealing the molecular principles of translational repression

Similar Modes of Interaction Enable Trailer Hitch and EDC3 To Associate with DCP1 and Me31B in Distinct Protein Complexes▿ †

Trailer Hitch (Tral or LSm15) and enhancer of decapping-3 (EDC3 or LSm16) are conserved eukaryotic members of the (L)Sm (Sm and Like-Sm) protein family. They have a similar domain organization, characterized by an N-terminal LSm domain and a central FDF motif; however, in Tral, the FDF motif is flanked by regions rich in charged residues, whereas in EDC3 the FDF motif is followed by a YjeF_N domain. We show that in Drosophila cells, Tral and EDC3 specifically interact with the decapping activator DCP1 and the DEAD-box helicase Me31B. Nevertheless, only Tral associates with the translational repressor CUP, whereas EDC3 associates with the decapping enzyme DCP2. Like EDC3, Tral interacts with DCP1 and localizes to mRNA processing bodies (P bodies) via the LSm domain. This domain remains monomeric in solution and adopts a divergent Sm fold that lacks the characteristic N-terminal α-helix, as determined by nuclear magnetic resonance analyses. Mutational analysis revealed that the structural integrity of the LSm domain is required for Tral both to interact with DCP1 and CUP and to localize to P-bodies. Furthermore, both Tral and EDC3 interact with the C-terminal RecA-like domain of Me31B through their FDF motifs. Together with previous studies, our results show that Tral and EDC3 are structurally related and use a similar mode to associate with common partners in distinct protein complexes

A Divergent Sm Fold in EDC3 Proteins Mediates DCP1 Binding and P-Body Targeting▿ †

Members of the (L)Sm (Sm and Sm-like) protein family are found across all kingdoms of life and play crucial roles in RNA metabolism. The P-body component EDC3 (enhancer of decapping 3) is a divergent member of this family that functions in mRNA decapping. EDC3 is composed of a N-terminal LSm domain, a central FDF domain, and a C-terminal YjeF-N domain. We show that this modular architecture enables EDC3 to interact with multiple components of the decapping machinery, including DCP1, DCP2, and Me31B. The LSm domain mediates DCP1 binding and P-body localization. We determined the three-dimensional structures of the LSm domains of Drosophila melanogaster and human EDC3 and show that the domain adopts a divergent Sm fold that lacks the characteristic N-terminal α-helix and has a disrupted β4-strand. This domain remains monomeric in solution and lacks several features that canonical (L)Sm domains require for binding RNA. The structures also revealed a conserved patch of surface residues that are required for the interaction with DCP1 but not for P-body localization. The conservation of surface and of critical structural residues indicates that LSm domains in EDC3 proteins adopt a similar fold that has separable novel functions that are absent in canonical (L)Sm proteins