Rapid Gene Evolution in an Ancient Post-transcriptional and Translational Regulatory System Compensates for Meiotic X Chromosomal Inactivation.

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.

Crystal structure and functional properties of the human CCR4-CAF1 deadenylase complex.

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.

4EHP and GIGYF1/2 Mediate Translation-Coupled Messenger RNA Decay.

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.

4E-T-bound mRNAs are stored in a silenced and deadenylated form.

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.

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 ß 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.

A low-complexity region in human XRN1 directly recruits deadenylation and decapping factors in 5'-3' messenger RNA decay.

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.

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.

A conserved CAF40-binding motif in metazoan NOT4 mediates association with the CCR4-NOT complex.

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.

Structural and biochemical analysis of a NOT1 MIF4G-like domain of the CCR4-NOT complex.

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.

Structural motifs in eIF4G and 4E-BPs modulate their binding to eIF4E to regulate translation initiation in yeast.

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.

Drosophila Bag-of-marbles directly interacts with the CAF40 subunit of the CCR4-NOT complex to elicit repression of mRNA targets.

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.

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression.

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.

A CAF40-binding motif facilitates recruitment of the CCR4-NOT complex to mRNAs targeted by Drosophila Roquin.

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.

The Structures of eIF4E-eIF4G Complexes Reveal an Extended Interface to Regulate Translation Initiation.

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.

Structure of the Dcp2-Dcp1 mRNA-decapping complex in the activated conformation.

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.

miRISC and the CCR4-NOT complex silence mRNA targets independently of 43S ribosomal scanning.

miRNAs associate with Argonaute (AGO) proteins to silence the expression of mRNA targets by inhibiting translation and promoting deadenylation, decapping, and mRNA degradation. A current model for silencing suggests that AGOs mediate these effects through the sequential recruitment of GW182 proteins, the CCR4-NOT deadenylase complex and the translational repressor and decapping activator DDX6. An alternative model posits that AGOs repress translation by interfering with eIF4A function during 43S ribosomal scanning and that this mechanism is independent of GW182 and the CCR4-NOT complex in Drosophila melanogaster Here, we show that miRNAs, AGOs, GW182, the CCR4-NOT complex, and DDX6/Me31B repress and degrade polyadenylated mRNA targets that are translated via scanning-independent mechanisms in both human and Dm cells. This and additional observations indicate a common mechanism used by these proteins and miRNAs to mediate silencing. This mechanism does not require eIF4A function during ribosomal scanning.

Distinct modes of recruitment of the CCR4-NOT complex by Drosophila and vertebrate Nanos.

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.

Mextli proteins use both canonical bipartite and novel tripartite binding modes to form eIF4E complexes that display differential sensitivity to 4E-BP regulation.

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.