Felix Tritschler

The GW182 protein family in animal cells: New insights into domains required for miRNA-mediated gene silencing

GW182 family proteins interact directly with Argonaute proteins and are required for miRNA-mediated gene silencing in animal cells. The domains of the GW182 proteins have recently been studied to determine their role in silencing. These studies revealed that the middle and C-terminal regions function as an autonomous domain with a repressive function that is independent of both the interaction with Argonaute proteins and of P-body localization. Such findings reinforce the idea that GW182 proteins are key components of miRNA repressor complexes in metazoa.

Structural Basis for the Mutually Exclusive Anchoring of P Body Components Edc3 and Tral to the Dead Box Protein Ddx6/Me31B.

The DEAD box helicase DDX6/Me31B functions in translational repression and mRNA decapping. How particular RNA helicases are recruited specifically to distinct functional complexes is poorly understood. We present the crystal structure of the DDX6 C-terminal RecA-like domain bound to a highly conserved FDF sequence motif in the decapping activator EDC3. The FDF peptide adopts an alpha-helical conformation upon binding to DDX6, occupying a shallow groove opposite to the DDX6 surface involved in RNA binding and ATP hydrolysis. Mutagenesis of Me31B shows the relevance of the FDF interaction surface both for Me31Bs accumulation in P bodies and for its ability to repress the expression of bound mRNAs. The translational repressor Tral contains a similar FDF motif. Together with mutational and competition studies, the structure reveals why the interactions of Me31B with EDC3 and Tral are mutually exclusive and how the respective decapping and translational repressor complexes might hook onto an mRNA substrate.

Crystal structure and ligand binding of the MID domain of a eukaryotic Argonaute protein

Argonaute (AGO) proteins are core components of RNA‐induced silencing complexes and have essential roles in RNA‐mediated gene silencing. They are characterized by a bilobal architecture, consisting of one lobe containing the amino‐terminal and PAZ domains and another containing the MID and PIWI domains. Except for the PAZ domain, structural information on eukaryotic AGO domains is not yet available. In this study, we report the crystal structure of the MID domain of the eukaryotic AGO protein QDE‐2 from Neurospora crassa. This domain adopts a Rossmann‐like fold and recognizes the 5′‐terminal nucleotide of a guide RNA in a manner similar to its prokaryotic counterparts. The 5′‐nucleotide‐binding site shares common residues with a second, adjacent ligand‐binding site, suggesting a mechanism for the cooperative binding of ligands to the MID domain of eukaryotic AGOs.

Role of GW182 proteins and PABPC1 in the miRNA pathway: a sense of déjà vu.

GW182 proteins have emerged as key components of microRNA (miRNA) silencing complexes in animals. Although the precise molecular function of GW182 proteins is not fully understood, new findings indicate that they act as poly(A)-binding protein (PABP)-interacting proteins (PAIPs) that promote gene silencing, at least in part, by interfering with cytoplasmic PABP1 (PABPC1) function during translation and mRNA stabilization. This recent discovery paves the way for future studies of miRNA silencing mechanisms.

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

ABSTRACT 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.

HPat provides a link between deadenylation and decapping in metazoa

A proline-rich region in the Drosophila Pat1 homologue works with the proteins C-terminal domain to recruit decapping and deadenylase complexes to target mRNAs.

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

ABSTRACT 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.

The RRM domain in GW182 proteins contributes to miRNA-mediated gene silencing

Proteins of the GW182 family interact with Argonaute proteins and are required for miRNA-mediated gene silencing. These proteins contain two structural domains, an ubiquitin-associated (UBA) domain and an RNA recognition motif (RRM), embedded in regions predicted to be unstructured. The structure of the RRM of Drosophila melanogaster GW182 reveals that this domain adopts an RRM fold, with an additional C-terminal α-helix. The helix lies on the β-sheet surface, generally used by these domains to bind RNA. This, together with the absence of aromatic residues in the conserved RNP1 and RNP2 motifs, and the lack of general affinity for RNA, suggests that the GW182 RRM does not bind RNA. The domain may rather engage in protein interactions through an unusual hydrophobic cleft exposed on the opposite face of the β-sheet. We further show that the GW182 RRM is dispensable for P-body localization and for interaction of GW182 with Argonaute-1 and miRNAs. Nevertheless, its deletion impairs the silencing activity of GW182 in a miRNA target-specific manner, indicating that this domain contributes to silencing. The conservation of structural and surface residues suggests that the RRM domain adopts a similar fold with a related function in insect and vertebrate GW182 family members.

DCP1 forms asymmetric trimers to assemble into active mRNA decapping complexes in metazoa

DCP1 stimulates the decapping enzyme DCP2, which removes the mRNA 5′ cap structure committing mRNAs to degradation. In multicellular eukaryotes, DCP1-DCP2 interaction is stabilized by additional proteins, including EDC4. However, most information on DCP2 activation stems from studies in S. cerevisiae, which lacks EDC4. Furthermore, DCP1 orthologs from multicellular eukaryotes have a C-terminal extension, absent in fungi. Here, we show that in metazoa, a conserved DCP1 C-terminal domain drives DCP1 trimerization. Crystal structures of the DCP1-trimerization domain reveal an antiparallel assembly comprised of three kinked α-helices. Trimerization is required for DCP1 to be incorporated into active decapping complexes and for efficient mRNA decapping in vivo. Our results reveal an unexpected connectivity and complexity of the mRNA decapping network in multicellular eukaryotes, which likely enhances opportunities for regulating mRNA degradation.

The C‐terminal α–α superhelix of Pat is required for mRNA decapping in metazoa

Pat proteins regulate the transition of mRNAs from a state that is translationally active to one that is repressed, committing targeted mRNAs to degradation. Pat proteins contain a conserved N‐terminal sequence, a proline‐rich region, a Mid domain and a C‐terminal domain (Pat‐C). We show that Pat‐C is essential for the interaction with mRNA decapping factors (i.e. DCP2, EDC4 and LSm1–7), whereas the P‐rich region and Mid domain have distinct functions in modulating these interactions. DCP2 and EDC4 binding is enhanced by the P‐rich region and does not require LSm1–7. LSm1–7 binding is assisted by the Mid domain and is reduced by the P‐rich region. Structural analysis revealed that Pat‐C folds into an α–α superhelix, exposing conserved and basic residues on one side of the domain. This conserved and basic surface is required for RNA, DCP2, EDC4 and LSm1–7 binding. The multiplicity of interactions mediated by Pat‐C suggests that certain of these interactions are mutually exclusive and, therefore, that Pat proteins switch decapping partners allowing transitions between sequential steps in the mRNA decapping pathway.