CCR4-NOT is a major effector complex in miRNAmediated gene silencing. It is recruited to miRNA targets through interactions with tryptophan (W)containing motifs in TNRC6/GW182 proteins and is required for both translational repression and degradation of miRNA targets. Here, we elucidate the structural basis for the repressive activity of CCR4-NOT and its interaction with TNRC6/GW182s. We show that the conserved CNOT9 subunit attaches to a domain of unknown function (DUF3819) in the CNOT1 scaffold. The resulting complex provides binding sites for TNRC6/GW182, and its crystal structure reveals tandem W-binding pockets located in CNOT9. We further show that the CNOT1 MIF4G domain interacts with the C-terminal RecA domain of DDX6, a translational repressor and decapping activator. The crystal structure of this complex demonstrates striking similarity to the eIF4G-eIF4A complex. Together, our data provide the missing physical links in a molecular pathway that connects miRNA target recognition with translational repression, deadenylation, and decapping.

The removal of the mRNA 5' cap structure by the decapping enzyme DCP2 leads to rapid 5'→3' mRNA degradation by XRN1, suggesting that the two processes are coordinated, but the coupling mechanism is unknown. DCP2 associates with the decapping activators EDC4 and DCP1. Here we show that XRN1 directly interacts with EDC4 and DCP1 in human and Drosophila melanogaster cells, respectively. In D. melanogaster cells, this interaction is mediated by the DCP1 EVH1 domain and a DCP1-binding motif (DBM) in the XRN1 C-terminal region. The NMR structure of the DCP1 EVH1 domain bound to the DBM reveals that the peptide docks at a conserved aromatic cleft, which is used by EVH1 domains to recognize proline-rich ligands. Our findings reveal a role for XRN1 in decapping and provide a molecular basis for the coupling of decapping to 5'→3' mRNA degradation.

The CCR4-NOT deadenylase complex is a master regulator of translation and mRNA stability. Its NOT module orchestrates recruitment of the catalytic subunits to target mRNAs. We report the crystal structure of the human NOT module formed by the CNOT1, CNOT2 and CNOT3 C-terminal (-C) regions. CNOT1-C provides a rigid scaffold consisting of two perpendicular stacks of HEAT-like repeats. CNOT2-C and CNOT3-C heterodimerize through their SH3-like NOT-box domains. The heterodimer is stabilized and tightly anchored to the surface of CNOT1 through an unexpected intertwined arrangement of peptide regions lacking defined secondary structure. These assembly peptides mold onto their respective binding surfaces and form extensive interfaces. Mutagenesis of individual interfaces and perturbation of endogenous protein ratios cause defects in complex assembly and mRNA decay. Our studies provide a structural framework for understanding the recruitment of the CCR4-NOT complex to mRNA targets.

We describe a data collection method that uses a single crystal to solve X-ray structures by native SAD (single-wavelength anomalous diffraction). We solved the structures of 11 real-life examples, including a human membrane protein, a protein-DNA complex and a 266-kDa multiprotein-ligand complex, using this method. The data collection strategy is suitable for routine structure determination and can be implemented at most macromolecular crystallography synchrotron beamlines.

Argonaute proteins (AGOs) are essential effectors in RNAmediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein.Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.P roteins of the Argonaute (AGO) family play essential roles in RNA-mediated gene silencing mechanisms in eukaryotes (1, 2). They are loaded with small noncoding RNAs to form the core of RNA-induced silencing complexes, which repress the expression of target genes at the transcriptional or posttranscriptional level (1, 2). The targets to be silenced are selected through base-pairing interactions between the loaded small RNA (also known as the guide RNA) and an mRNA target containing partially or fully complementary sequences (1-3).Thus far, structural information on full-length AGOs has been available only for the homologous proteins from Archaea and Eubacteria, which preferentially use DNA as a guide (4-10). These studies revealed that AGOs consist of four domains: the N-terminal domain; the PAZ domain, which binds the 3′ end of guide RNAs/DNAs; the MID domain, which provides a binding pocket for the 5′ phosphate of guide RNAs/DNAs; and the PIWI domain, which adopts an RNase H fold and has endonucleolytic activity in some, but not all, AGOs (4-11).For the eukaryotic AGO clade of Argonaute proteins, structural information is available only for the isolated PAZ domains of Drosophila melanogaster (Dm) AGO1 and AGO2, human AGO1 (12-16) and the MID domains of human AGO2 and Neurospora crassa (Nc) QDE-2 (17, 18). Structural information is also available for PAZ domains of the PIWI clade of AGOs (19,20). These studies showed that the PAZ and MID domains of eukaryotic AGOs adopt folds similar to the prokaryotic homologs and recognize the 3′-and 5′-terminal nucleotides of the guide strand, respectively, in a similar manner to their prokaryotic counterparts (12-18).Our previous structure of the isolate...