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The CCR4 and CAF1 deadenylases physically interact to form the CCR4-CAF1 complex and function as the catalytic core of the larger CCR4-NOT complex. Together, they are responsible for the eventual removal of the 3′-poly(A) tail from essentially all cellular mRNAs and consequently play a central role in the posttranscriptional regulation of gene expression. The individual properties of CCR4 and CAF1, however, and their respective contributions in different organisms and cellular environments are incompletely understood. Here, we determined the crystal structure of a human CCR4-CAF1 complex and characterized its enzymatic and substrate recognition properties. The structure reveals specific molecular details affecting RNA binding and hydrolysis, and confirms the CCR4 nuclease domain to be tethered flexibly with a considerable distance between both enzyme active sites. CCR4 and CAF1 sense nucleotide identity on both sides of the 3′-terminal phosphate, efficiently differentiating between single and consecutive non-A residues. In comparison to CCR4, CAF1 emerges as a surprisingly tunable enzyme, highly sensitive to pH, magnesium and zinc ions, and possibly allowing distinct reaction geometries. Our results support a picture of CAF1 as a primordial deadenylase, which gets assisted by CCR4 for better efficiency and by the assembled NOT proteins for selective mRNA targeting and regulation.

It is conventionally assumed that conserved pathways evolve slowly with little participation of gene evolution. Nevertheless, it has been recently observed that young genes can take over fundamental functions in essential biological processes, for example, development and reproduction. It is unclear how newly duplicated genes are integrated into ancestral networks and reshape the conserved pathways of important functions. Here, we investigated origination and function of two autosomal genes that evolved recently in Drosophila: Poseidon and Zeus, which were created by RNA-based duplications from the X-linked CAF40, a subunit of the conserved CCR4–NOT deadenylase complex involved in posttranscriptional and translational regulation. Knockdown and knockout assays show that the two genes quickly evolved critically important functions in viability and male fertility. Moreover, our transcriptome analysis demonstrates that the three genes have a broad and distinct effect in the expression of hundreds of genes, with almost half of the differentially expressed genes being perturbed exclusively by one paralog, but not the others. Co-immunoprecipitation and tethering assays show that the CAF40 paralog Poseidon maintains the ability to interact with the CCR4–NOT deadenylase complex and might act in posttranscriptional mRNA regulation. The rapid gene evolution in the ancient posttranscriptional and translational regulatory system may be driven by evolution of sex chromosomes to compensate for the meiotic X chromosomal inactivation (MXCI) in Drosophila.

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.

XRN1 is the major cytoplasmic exoribonuclease in eukaryotes, which degrades deadenylated and decapped mRNAs in the last step of the 5′–3′ mRNA decay pathway. Metazoan XRN1 interacts with decapping factors coupling the final stages of decay. Here, we reveal a direct interaction between XRN1 and the CCR4–NOT deadenylase complex mediated by a low-complexity region in XRN1, which we term the ‘C-terminal interacting region’ or CIR. The CIR represses reporter mRNA deadenylation in human cells when overexpressed and inhibits CCR4–NOT and isolated CAF1 deadenylase activity in vitro. Through complementation studies in an XRN1-null cell line, we dissect the specific contributions of XRN1 domains and regions toward decay of an mRNA reporter. We observe that XRN1 binding to the decapping activator EDC4 counteracts the dominant negative effect of CIR overexpression on decay. Another decapping activator PatL1 directly interacts with CIR and alleviates the CIR-mediated inhibition of CCR4–NOT activity in vitro. Ribosome profiling revealed that XRN1 loss impacts not only on mRNA levels but also on the translational efficiency of many cellular transcripts likely as a consequence of incomplete decay. Our findings reveal an additional layer of direct interactions in a tightly integrated network of factors mediating deadenylation, decapping and 5′–3′ exonucleolytic decay.

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.

The multisubunit CCR4–NOT mRNA deadenylase complex plays important roles in the posttranscriptional regulation of gene expression. The NOT4 E3 ubiquitin ligase is a stable component of the CCR4–NOT complex in yeast but does not copurify with the human or Drosophila melanogaster complex. Here we show that the C-terminal regions of human and D. melanogaster NOT4 contain a conserved sequence motif that directly binds the CAF40 subunit of the CCR4–NOT complex (CAF40-binding motif [CBM]). In addition, nonconserved sequences flanking the CBM also contact other subunits of the complex. Crystal structures of the CBM–CAF40 complex reveal a mutually exclusive binding surface for NOT4 and Roquin or Bag of marbles mRNA regulatory proteins. Furthermore, CAF40 depletion or structure-guided mutagenesis to disrupt the NOT4–CAF40 interaction impairs the ability of NOT4 to elicit decay of tethered reporter mRNAs in cells. Together with additional sequence analyses, our results reveal the molecular basis for the association of metazoan NOT4 with the CCR4–NOT complex and show that it deviates substantially from yeast. They mark the NOT4 ubiquitin ligase as an ancient but nonconstitutive cofactor of the CCR4–NOT deadenylase with potential recruitment and/or effector functions.

Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that the 4EHP–GIGYF1/2 complexes trigger co-translational mRNA decay as a result of perturbed elongation. Human cells lacking 4EHP and GIGYF1/2 proteins accumulate transcripts known to be degraded in a translation-dependent manner or with prominent ribosome pausing. These include among others, mRNAs encoding secretory and membrane-bound proteins or α- and β-tubulin subunits. In addition, 4EHP–GIGYF1/2 complexes fail to reduce target mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the mRNA cap structure, DDX6 or GYF domain-associated factors. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.

Human 4E-T is an eIF4E-binding protein (4E-BP) present in processing (P)-bodies that represses translation and regulates decay of mRNAs destabilized by AU-rich elements and microRNAs (miRNAs). However, the underlying regulatory mechanisms are still unclear. Here, we show that upon mRNA binding 4E-T represses translation and promotes deadenylation via the recruitment of the CCR4–NOT deadenylase complex. The interaction with CCR4–NOT is mediated by previously uncharacterized sites in the middle region of 4E-T. Importantly, mRNA decapping and decay are inhibited by 4E-T and the deadenylated target is stored in a repressed form. Inhibition of mRNA decapping requires the interaction of 4E-T with the cap-binding proteins eIF4E/4EHP. We further show that regulation of decapping by 4E-T participates in mRNA repression by the miRNA effector protein TNRC6B and that 4E-T overexpression interferes with tristetraprolin (TTP)- and NOT1-mediated mRNA decay. Thus, we postulate that 4E-T modulates 5′-to-3′ decay by swapping the fate of a deadenylated mRNA from complete degradation to storage. Our results provide insight into the mechanism of mRNA storage that controls localized translation and mRNA stability in P-bodies.

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine–tyrosine–phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP–GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity.

Eukaryotic initiation factor 4G (eIF4G) plays a central role in translation initiation through its interactions with the cap-binding protein eIF4E. This interaction is a major drug target for repressing translation and is naturally regulated by 4E-binding proteins (4E-BPs). 4E-BPs and eIF4G compete for binding to the eIF4E dorsal surface via a shared canonical 4E-binding motif, but also contain auxiliary eIF4E-binding sequences, which were assumed to contact non-overlapping eIF4E surfaces. However, it is unknown how metazoan eIF4G auxiliary sequences bind eIF4E. Here, we describe crystal structures of human and Drosophila melanogaster eIF4E-eIF4G complexes, which unexpectedly reveal that the eIF4G auxiliary sequences bind to the lateral surface of eIF4E, using a similar mode to that of 4E-BPs. Our studies provide a molecular model of the eIF4E-eIF4G complex, shed light on the competition mechanism of 4E-BPs, and enable the rational design of selective eIF4G inhibitors to dampen dysregulated translation in disease.

The removal of the mRNA 5' cap (decapping) by Dcp2 shuts down translation and commits mRNA to full degradation. Dcp2 activity is enhanced by activator proteins such as Dcp1 and Edc1. However, owing to conformational flexibility, the active conformation of Dcp2 and the mechanism of decapping activation have remained unknown. Here, we report a 1.6-Å-resolution crystal structure of the Schizosaccharomyces pombe Dcp2-Dcp1 heterodimer in an unprecedented conformation that is tied together by an intrinsically disordered peptide from Edc1. In this ternary complex, an unforeseen rotation of the Dcp2 catalytic domain allows residues from both Dcp2 and Dcp1 to cooperate in RNA binding, thus explaining decapping activation by increased substrate affinity. The architecture of the Dcp2-Dcp1-Edc1 complex provides a rationale for the conservation of a sequence motif in Edc1 that is also present in unrelated decapping activators, thus indicating that the presently described mechanism of decapping activation is evolutionarily conserved.

Nanos proteins repress the expression of target mRNAs by recruiting effector complexes through non‐conserved N‐terminal regions. In vertebrates, Nanos proteins interact with the NOT1 subunit of the CCR4–NOT effector complex through a NOT1 interacting motif (NIM), which is absent in Nanos orthologs from several invertebrate species. Therefore, it has remained unclear whether the Nanos repressive mechanism is conserved and whether it also involves direct interactions with the CCR4–NOT deadenylase complex in invertebrates. Here, we identify an effector domain (NED) that is necessary for the Drosophila melanogaster (Dm) Nanos to repress mRNA targets. The NED recruits the CCR4–NOT complex through multiple and redundant binding sites, including a central region that interacts with the NOT module, which comprises the C‐terminal domains of NOT1–3. The crystal structure of the NED central region bound to the NOT module reveals an unanticipated bipartite binding interface that contacts NOT1 and NOT3 and is distinct from the NIM of vertebrate Nanos. Thus, despite the absence of sequence conservation, the N‐terminal regions of Nanos proteins recruit CCR4–NOT to assemble analogous repressive complexes.

Current models of mRNA turnover indicate that cytoplasmic degradation is coupled with translation. However, our understanding of the molecular events that coordinate ribosome transit with the mRNA decay machinery is still limited. Here, we show that 4EHP-GIGYF1/2 complexes trigger co-translational mRNA decay. Human cells lacking these proteins accumulate mRNAs with prominent ribosome pausing. They include, among others, transcripts encoding secretory and membrane-bound proteins or tubulin subunits. In addition, 4EHP-GIGYF1/2 complexes fail to reduce mRNA levels in the absence of ribosome stalling or upon disruption of their interaction with the cap structure, DDX6, and ZNF598. We further find that co-translational binding of GIGYF1/2 to the mRNA marks transcripts with perturbed elongation to decay. Our studies reveal how a repressor complex linked to neurological disorders minimizes the protein output of a subset of mRNAs.

The CCR4-NOT complex plays a central role in the regulation of gene expression and degradation of messenger RNAs. The multisubunit complex assembles on the NOT1 protein, which acts as a 'scaffold' and is highly conserved in eukaryotes. NOT1 consists of a series of helical domains that serve as docking sites for other CCR4-NOT subunits. We describe a crystal structure of a connector domain of NOT1 from the thermophilic fungus Chaetomium thermophilum (Ct). Comparative structural analysis indicates that this domain adopts a MIF4G-like fold and we have termed it the MIF4G-C domain. Solution scattering studies indicate that the human MIF4G-C domain likely adopts a very similar fold to the Ct MIF4G-C. MIF4G domains have been described to mediate interactions with DEAD-box helicases such as DDX6. However, comparison of the interfaces of the MIF4G-C with the MIF4G domain of NOT1 that interacts with DDX6 reveals key structural differences that explain why the MIF4G-C does not bind DDX6. We further show that the human MIF4G-C does not interact stably with other subunits of the CCR4-NOT complex. The structural conservation of the MIF4G-C domain suggests that it may have an important but presently undefined role in the CCR4-NOT complex.

Drosophila melanogaster Bag-of-marbles (Bam) promotes germline stem cell (GSC) differentiation by repressing the expression of mRNAs encoding stem cell maintenance factors. Bam interacts with Benign gonial cell neoplasm (Bgcn) and the CCR4 deadenylase, a catalytic subunit of the CCR4–NOT complex. Bam has been proposed to bind CCR4 and displace it from the CCR4–NOT complex. Here, we investigated the interaction of Bam with the CCR4–NOT complex by using purified recombinant proteins. Unexpectedly, we found that Bam does not interact with CCR4 directly but instead binds to the CAF40 subunit of the complex in a manner mediated by a conserved N-terminal CAF40-binding motif (CBM). The crystal structure of the Bam CBM bound to CAF40 reveals that the CBM peptide adopts an α-helical conformation after binding to the concave surface of the crescent-shaped CAF40 protein. We further show that Bam-mediated mRNA decay and translational repression depend entirely on Bam's interaction with CAF40. Thus, Bam regulates the expression of its mRNA targets by recruiting the CCR4–NOT complex through interaction with CAF40.

Nucleic acids symposium series 46(13), 6893 - 6908 (2018). doi:10.1093/nar/gky542, The interaction of the eukaryotic initiation factor 4G (eIF4G) with the cap-binding protein eIF4E initiates cap-dependent translation and is regulated by the 4E-binding proteins (4E-BPs), which compete with eIF4G to repress translation. Metazoan eIF4G and 4E-BPs interact with eIF4E via canonical and non-canonical motifs that bind to the dorsal and lateral surface of eIF4E in a bipartite recognition mode. However, previous studies pointed to mechanistic differences in how fungi and metazoans regulate protein synthesis. We present crystal structures of the yeast eIF4E bound to two yeast 4E-BPs, p20 and Eap1p, as well as crystal structures of a fungal eIF4E–eIF4G complex. We demonstrate that the core principles of molecular recognition of eIF4E are in fact highly conserved among translational activators and repressors in eukaryotes. Finally, we reveal that highly specialized structural motifs do exist and serve to modulate the affinity of protein-protein interactions that regulate cap-dependent translation initiation in fungi., Published by Oxford Univ. Press, Oxford

MicroRNAs (miRNAs) are a conserved class of small non-coding RNAs that assemble with Argonaute proteins into miRNA-induced silencing complexes (miRISCs) to direct post-transcriptional silencing of complementary mRNA targets. Silencing is accomplished through a combination of translational repression and mRNA destabilization, with the latter contributing to most of the steady-state repression in animal cell cultures. Degradation of the mRNA target is initiated by deadenylation, which is followed by decapping and 5'-to-3' exonucleolytic decay. Recent work has enhanced our understanding of the mechanisms of silencing, making it possible to describe in molecular terms a continuum of direct interactions from miRNA target recognition to mRNA deadenylation, decapping and 5'-to-3' degradation. Furthermore, an intricate interplay between translational repression and mRNA degradation is emerging.

The eIF4E-binding proteins (4E-BPs) represent a diverse class of translation inhibitors that are often deregulated in cancer cells. 4E-BPs inhibit translation by competing with eIF4G for binding to eIF4E through an interface that consists of canonical and non-canonical eIF4E-binding motifs connected by a linker. The lack of high-resolution structures including the linkers, which contain phosphorylation sites, limits our understanding of how phosphorylation inhibits complex formation. Furthermore, the binding mechanism of the non-canonical motifs is poorly understood. Here, we present structures of human eIF4E bound to 4E-BP1 and fly eIF4E bound to Thor, 4E-T, and eIF4G. These structures reveal architectural elements that are unique to 4E-BPs and provide insight into the consequences of phosphorylation. Guided by these structures, we designed and crystallized a 4E-BP mimic that shows increased repressive activity. Our studies pave the way for the rational design of 4E-BP mimics as therapeutic tools to decrease translation during oncogenic transformation.

CCR4-NOT is a major effector complex in miRNA-mediated 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 RNA-binding proteins of the Nanos family play an essential role in germ cell development and survival in a wide range of metazoan species. They function by suppressing the expression of target mRNAs through the recruitment of effector complexes, which include the CCR4–NOT deadenylase complex. Here, we show that the three human Nanos paralogs (Nanos1–3) interact with the CNOT1 C-terminal domain and determine the structural basis for the specific molecular recognition. Nanos1–3 bind CNOT1 through a short CNOT1-interacting motif (NIM) that is conserved in all vertebrates and some invertebrate species. The crystal structure of the human Nanos1 NIM peptide bound to CNOT1 reveals that the peptide opens a conserved hydrophobic pocket on the CNOT1 surface by inserting conserved aromatic residues. The substitutions of these aromatic residues in the Nanos1–3 NIMs abolish binding to CNOT1 and abrogate the ability of the proteins to repress translation. Our findings provide the structural basis for the recruitment of the CCR4–NOT complex by vertebrate Nanos, indicate that the NIMs are the major determinants of the translational repression mediated by Nanos, and identify the CCR4–NOT complex as the main effector complex for Nanos function.

The removal of the 5′-cap structure by the decapping enzyme DCP2 and its coactivator DCP1 shuts down translation and exposes the mRNA to 5′-to-3′ exonucleolytic degradation by XRN1. Although yeast DCP1 and DCP2 directly interact, an additional factor, EDC4, promotes DCP1–DCP2 association in metazoan. Here, we elucidate how the human proteins interact to assemble an active decapping complex and how decapped mRNAs are handed over to XRN1. We show that EDC4 serves as a scaffold for complex assembly, providing binding sites for DCP1, DCP2 and XRN1. DCP2 and XRN1 bind simultaneously to the EDC4 C-terminal domain through short linear motifs (SLiMs). Additionally, DCP1 and DCP2 form direct but weak interactions that are facilitated by EDC4. Mutational and functional studies indicate that the docking of DCP1 and DCP2 on the EDC4 scaffold is a critical step for mRNA decapping in vivo. They also revealed a crucial role for a conserved asparagine–arginine containing loop (the NR-loop) in the DCP1 EVH1 domain in DCP2 activation. Our data indicate that DCP2 activation by DCP1 occurs preferentially on the EDC4 scaffold, which may serve to couple DCP2 activation by DCP1 with 5′-to-3′ mRNA degradation by XRN1 in human cells.

MicroRNAs (miRNAs) are small, ~22-nucleotide-long noncoding RNAs. They silence the expression of messenger RNAs (mRNAs) containing complementary sequences ( 1 ). The human genome encodes ~1500 miRNAs, each with the potential to bind hundreds of different mRNAs ( 1 ). miRNAs regulate many biological processes, and the dysregulation of their expression is linked to various human diseases, including cancer ( 1 ). To exert their repressive function, miRNAs associate with the Argonaute family of proteins (AGOs) to form the core of miRNA-induced silencing complexes (miRISCs) ( 1 ) (see the figure). In animals, miRISCs silence mRNA expression at two levels, by preventing protein production (translation) and inducing mRNA degradation. Over the past decade, progress has been made in our understanding of the mechanism by which miRISCs induce mRNA degradation, but the question of how miRISCs repress translation remains elusive.

The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 adaptor protein crystal structure that, unexpectedly, forms intertwined and asymmetric homodimers. Dimerization is mediated by a coiled coil that links an N-terminal pseudokinase to a C-terminal knob domain. The PAN3 pseudokinase binds ATP, and this function is required for mRNA degradation in vivo. We further identified conserved surfaces required for mRNA degradation, including the binding surface for the PAN2 deadenylase on the knob domain. The most remarkable structural feature is the presence of a tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in human cells. Together, our data reveal the structural basis for the interaction of PAN3 with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6 proteins.

MicroRNA (miRNA)-induced silencing complexes (miRISCs) repress translation and promote degradation of miRNA targets. Target degradation occurs through the 5'-to-3' messenger RNA (mRNA) decay pathway, wherein, after shortening of the mRNA poly(A) tail, the removal of the 5' cap structure by decapping triggers irreversible decay of the mRNA body. Here, we demonstrate that miRISC enhances the association of the decapping activators DCP1, Me31B and HPat with deadenylated miRNA targets that accumulate when decapping is blocked. DCP1 and Me31B recruitment by miRISC occurs before the completion of deadenylation. Remarkably, miRISC recruits DCP1, Me31B and HPat to engineered miRNA targets transcribed by RNA polymerase III, which lack a cap structure, a protein-coding region and a poly(A) tail. Furthermore, miRISC can trigger decapping and the subsequent degradation of mRNA targets independently of ongoing deadenylation. Thus, miRISC increases the local concentration of the decapping machinery on miRNA targets to facilitate decapping and irreversibly shut down their translation.

The CCR4-NOT complex plays a crucial role in post-transcriptional mRNA regulation in eukaryotes. This complex catalyzes the removal of mRNA poly(A) tails, thereby repressing translation and committing an mRNA to degradation. The conserved core of the complex is assembled by the interaction of at least two modules: the NOT module, which minimally consists of NOT1, NOT2 and NOT3, and a catalytic module comprising two deadenylases, CCR4 and POP2/CAF1. Additional complex subunits include CAF40 and two newly identified human subunits, NOT10 and C2orf29. The role of the NOT10 and C2orf29 subunits and how they are integrated into the complex are unknown. Here, we show that the Drosophila melanogaster NOT10 and C2orf29 orthologs form a complex that interacts with the N-terminal domain of NOT1 through C2orf29. These interactions are conserved in human cells, indicating that NOT10 and C2orf29 define a conserved module of the CCR4-NOT complex. We further investigated the assembly of the D. melanogaster CCR4-NOT complex, and demonstrate that the conserved armadillo repeat domain of CAF40 interacts with a region of NOT1, comprising a domain of unknown function, DUF3819. Using tethering assays, we show that each subunit of the CCR4-NOT complex causes translational repression of an unadenylated mRNA reporter and deadenylation and degradation of a polyadenylated reporter. Therefore, the recruitment of a single subunit of the complex to an mRNA target induces the assembly of the complete CCR4-NOT complex, resulting in a similar regulatory outcome.

microRNAs (miRNAs) silence gene expression by suppressing protein production and/or by promoting mRNA decay. To elucidate how silencing is accomplished, we screened an RNA interference library for suppressors of miRNA-mediated regulation in Drosophila melanogaster cells. In addition to proteins known to be required for miRNA biogenesis and function (i.e., Drosha, Pasha, Dicer-1, AGO1, and GW182), the screen identified the decapping activator Ge-1 as being required for silencing by miRNAs. Depleting Ge-1 alone and/or in combination with other decapping activators (e.g., DCP1, EDC3, HPat, or Me31B) suppresses silencing of several miRNA targets, indicating that miRNAs elicit mRNA decapping. A comparison of gene expression profiles in cells depleted of AGO1 or of individual decapping activators shows that ∼15% of AGO1-targets are also regulated by Ge-1, DCP1, and HPat, whereas 5% are dependent on EDC3 and LSm1–7. These percentages are underestimated because decapping activators are partially redundant. Furthermore, in the absence of active translation, some miRNA targets are stabilized, whereas others continue to be degraded in a miRNA-dependent manner. These findings suggest that miRNAs mediate post-transcriptional gene silencing by more than one mechanism.

MicroRNAs (miRNAs) silence the expression of target genes post-transcriptionally. Their function is mediated by the Argonaute proteins (AGOs), which colocalize to P-bodies with mRNA degradation enzymes. Mammalian P-bodies are also marked by the GW182 protein, which interacts with the AGOs and is required for miRNA function. We show that depletion of GW182 leads to changes in mRNA expression profiles strikingly similar to those observed in cells depleted of the essential Drosophila miRNA effector AGO1, indicating that GW182 functions in the miRNA pathway. When GW182 is bound to a reporter transcript, it silences its expression, bypassing the requirement for AGO1. Silencing by GW182 is effected by changes in protein expression and mRNA stability. Similarly, miRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay, and both mechanisms require GW182. mRNA degradation, but not translational repression, by GW182 or miRNAs is inhibited in cells depleted of CAF1, NOT1, or the decapping DCP1:DCP2 complex. We further show that the N-terminal GW repeats of GW182 interact with the PIWI domain of AGO1. Our findings indicate that GW182 links the miRNA pathway to mRNA degradation by interacting with AGO1 and promoting decay of at least a subset of miRNA targets.

Human (Hs) Roquin1 and Roquin2 are RNA-binding proteins that promote mRNA target degradation through the recruitment of the CCR4-NOT deadenylase complex and are implicated in the prevention of autoimmunity. Roquin1 recruits CCR4-NOT via a C-terminal region that is not conserved in Roquin2 or in invertebrate Roquin. Here we show that Roquin2 and Drosophila melanogaster (Dm) Roquin also interact with the CCR4-NOT complex through their C-terminal regions. The C-terminal region of Dm Roquin contains multiple motifs that mediate CCR4-NOT binding. One motif binds to the CAF40 subunit of the CCR4-NOT complex. The crystal structure of the Dm Roquin CAF40-binding motif (CBM) bound to CAF40 reveals that the CBM adopts an α-helical conformation upon binding to a conserved surface of CAF40. Thus, despite the lack of sequence conservation, the C-terminal regions of Roquin proteins act as an effector domain that represses the expression of mRNA targets via recruitment of the CCR4-NOT complex., Roquin proteins downregulate target mRNA expression by recruiting effectors such as the CCR4-NOT deadenylase complex. Here the authors provide molecular details of how Roquin proteins recruit the CCR4-NOT complex to repress the expression of its targets.

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 Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5′ cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.

Despite their widespread roles as regulators of gene expression, important questions remain about target regulation by microRNAs. Animal microRNAs were originally thought to repress target translation, with little or no influence on mRNA abundance, whereas the reverse was thought to be true in plants. Now, however, it is clear that microRNAs can induce mRNA degradation in animals and, conversely, translational repression in plants. Recent studies have made important advances in elucidating the relative contributions of these two different modes of target regulation by microRNAs. They have also shed light on the specific mechanisms of target silencing, which, although it differs fundamentally between plants and animals, shares some common features between the two kingdoms.

Two PABPC1-binding sites in GW182 proteins promote miRNA-mediated gene silencing Previous studies have suggested that the mechanism of miRNA-mediated silencing may differ between human and Drosophila cells. Here, a direct comparison demonstrates that the mechanism is conserved and the GW182–PABP interaction is required for silencing in vivo., miRNA-mediated gene silencing requires the GW182 proteins, which are characterized by an N-terminal domain that interacts with Argonaute proteins (AGOs), and a C-terminal silencing domain (SD). In Drosophila melanogaster (Dm) GW182 and a human (Hs) orthologue, TNRC6C, the SD was previously shown to interact with the cytoplasmic poly(A)-binding protein (PABPC1). Here, we show that two regions of GW182 proteins interact with PABPC1: the first contains a PABP-interacting motif 2 (PAM2; as shown before for TNRC6C) and the second contains the M2 and C-terminal sequences in the SD. The latter mediates indirect binding to the PABPC1 N-terminal domain. In D. melanogaster cells, the second binding site dominates; however, in HsTNRC6A–C the PAM2 motif is essential for binding to both Hs and DmPABPC1. Accordingly, a single amino acid substitution in the TNRC6A–C PAM2 motif abolishes the interaction with PABPC1. This mutation also impairs TNRC6s silencing activity. Our findings reveal that despite species-specific differences in the relative strength of the PABPC1-binding sites, the interaction between GW182 proteins and PABPC1 is critical for miRNA-mediated silencing in animal cells.

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that detects and degrades mRNAs containing premature stop codons (PTCs). In vertebrates, PTCs trigger efficient NMD when located upstream of an exon junction complex (EJC). Degradation of PTC-containing mRNAs requires the endonucleolytic activity of SMG6, a conserved NMD factor; nevertheless, the precise role for the EJC in NMD and how the SMG6 endonuclease is recruited to NMD targets have been unclear. Here we show that SMG6 interacts directly with the EJC via two conserved EJC-binding motifs (EBMs). We further show that the SMG6–EJC interaction is required for NMD. Our results reveal an unprecedented role for the EJC in recruiting the SMG6 endonuclease to NMD targets. More generally, our findings identify the EBM as a protein motif present in a handful of proteins, and suggest that EJCs establish multiple and mutually exclusive interactions with various protein partners, providing a plausible explanation for the myriad functions performed by this complex in post-transcriptional mRNA regulation.

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.

A proline-rich region in the Drosophila Pat1 homologue works with the protein's C-terminal domain to recruit decapping and deadenylase complexes to target mRNAs., Decapping of eukaryotic messenger RNAs (mRNAs) occurs after they have undergone deadenylation, but how these processes are coordinated is poorly understood. In this study, we report that Drosophila melanogaster HPat (homologue of Pat1), a conserved decapping activator, interacts with additional decapping factors (e.g., Me31B, the LSm1–7 complex, and the decapping enzyme DCP2) and with components of the CCR4–NOT deadenylase complex. Accordingly, HPat triggers deadenylation and decapping when artificially tethered to an mRNA reporter. These activities reside, unexpectedly, in a proline-rich region. However, this region alone cannot restore decapping in cells depleted of endogenous HPat but also requires the middle (Mid) and the very C-terminal domains of HPat. We further show that the Mid and C-terminal domains mediate HPat recruitment to target mRNAs. Our results reveal an unprecedented role for the proline-rich region and the C-terminal domain of metazoan HPat in mRNA decapping and suggest that HPat is a component of the cellular mechanism that couples decapping to deadenylation in vivo.

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.

GW182 family proteins are essential in animal cells for microRNA (miRNA)-mediated gene silencing, yet the molecular mechanism that allows GW182 to promote translational repression and mRNA decay remains largely unknown. Previous studies showed that while the GW182 N-terminal domain interacts with Argonaute proteins, translational repression and degradation of miRNA targets are promoted by a bipartite silencing domain comprising the GW182 middle and C-terminal regions. Here we show that the GW182 C-terminal region is required for GW182 to release silenced mRNPs; moreover, GW182 dissociates from miRNA targets at a step of silencing downstream of deadenylation, indicating that GW182 is required to initiate but not to maintain silencing. In addition, we show that the GW182 bipartite silencing domain competes with eukaryotic initiation factor 4G for binding to PABPC1. The GW182-PABPC1 interaction is also required for miRNA target degradation; accordingly, we observed that PABPC1 associates with components of the CCR4-NOT deadenylase complex. Finally, we show that PABPC1 overexpression suppresses the silencing of miRNA targets. We propose a model in which the GW182 silencing domain promotes translational repression, at least in part, by interfering with mRNA circularization and also recruits the deadenylase complex through the interaction with PABPC1.

The nonsense-mediated mRNA decay (NMD) pathway promotes rapid degradation of mRNAs containing premature translation termination codons (PTCs or nonsense codons), preventing accumulation of potentially detrimental truncated proteins. In metazoa, seven genes (upf1, upf2, upf3, smg1, smg5, smg6, and smg7) have been identified as essential for NMD; here we show that the zebrafish genome encodes orthologs of upf1, upf2, smg1, and smg5 to smg7 and two upf3 paralogs. We also show that Upf1 is required for degradation of PTC-containing mRNAs in zebrafish embryos. Moreover, its depletion has a severe impact on embryonic development, early patterning, and viability. Similar phenotypes are observed in Upf2-, Smg5-, or Smg6-depleted embryos, suggesting that zebrafish embryogenesis requires an active NMD pathway. Using cultured cells, we demonstrate that the ability of a PTC to trigger NMD is strongly stimulated by downstream exon-exon boundaries. Thus, as in mammals and plants but in contrast to invertebrates and fungi, NMD is coupled to splicing in zebrafish. Our results together with previous studies show that NMD effectors are essential for vertebrate embryogenesis and suggest that the coupling of splicing and NMD has been maintained in vertebrates but lost in fungi and invertebrates.

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.

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 alpha-helix. The helix lies on the beta-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 beta-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.

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 Me31B's 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.

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.

MicroRNAs (miRNAs) silence gene expression by binding 3' untranslated regions of target mRNAs. Recent studies suggested silencing is achieved through either recruitment of eIF6, which prevents ribosome assembly, or displacement of eIF4E from the mRNA 5' cap structure. Using Drosophila melanogaster cells, we show that eIF6 is not required for silencing. In contrast, silencing is abolished by mutating Argonaute 1 (AGO1) at two conserved phenylalanine residues predicted to mediate binding to the cap structure. Notably, we found these mutations also prevented AGO1 from interacting with GW182 and miRNAs, indicating that the essential role of these residues is unrelated to cap binding. Consistently, depleting GW182 or overexpressing its AGO1 binding domain relieved silencing of all reporters tested, including those lacking a poly(A) tail. Together, our findings show that miRNA function is effected by AGO1-GW182 complexes and the role of GW182 in silencing goes beyond promoting deadenylation.

P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.

Post-transcriptional processes have a central role in the regulation of eukaryotic gene expression. Although it has been known for a long time that these processes are functionally linked, often by the use of common protein factors, it has only recently become apparent that many of these processes are also physically connected. Indeed, proteins that are involved in mRNA degradation, translational repression, mRNA surveillance and RNA-mediated gene silencing, together with their mRNA targets, colocalize within discrete cytoplasmic domains known as P bodies. The available evidence indicates that P bodies are sites where mRNAs that are not being translated accumulate, the information carried by associated proteins and regulatory RNAs is integrated, and their fate - either translation, silencing or decay - is decided.

The eIF4E-binding proteins (4E-BPs) are a diverse class of translation regulators that share a canonical eIF4E-binding motif (4E-BM) with eIF4G. Consequently, they compete with eIF4G for binding to eIF4E, thereby inhibiting translation initiation. Mextli (Mxt) is an unusual 4E-BP that promotes translation by also interacting with eIF3. Here we present the crystal structures of the eIF4E-binding regions of the Drosophila melanogaster (Dm) and Caenorhabditis elegans (Ce) Mxt proteins in complex with eIF4E in the cap-bound and cap-free states. The structures reveal unexpected evolutionary plasticity in the eIF4E-binding mode, with a classical bipartite interface for Ce Mxt and a novel tripartite interface for Dm Mxt. Both interfaces comprise a canonical helix and a noncanonical helix that engage the dorsal and lateral surfaces of eIF4E, respectively. Remarkably, Dm Mxt contains a C-terminal auxiliary helix that lies anti-parallel to the canonical helix on the eIF4E dorsal surface. In contrast to the eIF4G and Ce Mxt complexes, the Dm eIF4E–Mxt complexes are resistant to competition by bipartite 4E-BPs, suggesting that Dm Mxt can bind eIF4E when eIF4G binding is inhibited. Our results uncovered unexpected diversity in the binding modes of 4E-BPs, resulting in eIF4E complexes that display differential sensitivity to 4E-BP regulation.

Recent genome-wide identification of nonsense-mediated mRNA decay (NMD) targets in yeast, fruitfly and human cells has provided insight into the biological functions and evolution of this mRNA quality control mechanism, revealing that NMD post-transcriptionally regulates an important fraction of the transcriptome. NMD targets are associated with a broad range of biological processes, but most of these targets are not encoded by orthologous genes across different species. Yeast and fruitfly NMD effectors regulate common targets in concert, but parallel pathways have evolved in humans, whereby NMD effectors have acquired additional functions. Thus, the phenotypic differences observed across species after inhibition of NMD are driven not only by the functional diversification of NMD effectors but also by changes in the repertoire of regulated genes.