Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

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.

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.

Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation.

The Alu element is the most successful human genomic parasite affecting development and causing disease. It originated as a retrotransposon during early primate evolution of the gene encoding the signal recognition particle (SRP) RNA. We defined a minimal Alu RNA sufficient for effective retrotransposition and determined a high-resolution structure of its complex with the SRP9/14 proteins. The RNA adopts a compact, closed conformation that matches the envelope of the SRP Alu domain in the ribosomal translation elongation factor-binding site. Conserved structural elements in SRP RNAs support an ancient function of the closed conformation that predates SRP9/14. Structure-based mutagenesis shows that retrotransposition requires the closed conformation of the Alu ribonucleoprotein particle and is consistent with the recognition of stalled ribosomes. We propose that ribosome stalling is a common cause for the cis-preference of the mammalian L1 retrotransposon and for the efficiency of the Alu RNA in hijacking nascent L1 reverse transcriptase.

Molecular architecture of 4E-BP translational inhibitors bound to eIF4E.

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.

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.

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.

A DDX6-CNOT1 complex and W-binding pockets in CNOT9 reveal direct links between miRNA target recognition and silencing.

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.

Structural basis for the Nanos-mediated recruitment of the CCR4-NOT complex and translational repression.

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.

Structure of the PAN3 pseudokinase reveals the basis for interactions with the PAN2 deadenylase and the GW182 proteins.

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.

An unusual arrangement of two 14-3-3-like domains in the SMG5-SMG7 heterodimer is required for efficient nonsense-mediated mRNA decay.

The nonsense-mediated mRNA decay (NMD) pathway triggers the rapid degradation of aberrant mRNAs containing premature translation termination codons (PTCs). In metazoans, NMD requires three 14-3-3-like proteins: SMG5, SMG6, and SMG7. These proteins are recruited to PTC-containing mRNAs through the interaction of their 14-3-3-like domains with phosphorylated UPF1, the central NMD effector. Recruitment of SMG5, SMG6, and SMG7 causes NMD target degradation. In this study, we report the crystal structure of the Caenorhabditis elegans SMG5-SMG7 complex. The 14-3-3-like phosphopeptide recognition domains of SMG5 and SMG7 heterodimerize in an unusual perpendicular back-to-back orientation in which the peptide-binding sites face opposite directions. Structure-based mutants and functional assays indicate that the SMG5-SMG7 interaction is conserved and is crucial for efficient NMD in human cells. Notably, we demonstrate that heterodimerization increases the affinity of the SMG5-SMG7 complex for UPF1. Furthermore, we show that the degradative activity of the SMG5-SMG7 complex resides in SMG7 and that the SMG5-SMG7 complex and SMG6 play partially redundant roles in the degradation of aberrant mRNAs. We propose that the SMG5-SMG7 complex binds to phosphorylated UPF1 with high affinity and recruits decay factors to the mRNA target through SMG7, thus promoting target degradation.

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.

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.

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.

Fast native-SAD phasing for routine macromolecular structure determination.

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.

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 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.

Structure and properties of the esterase from non-LTR retrotransposons suggest a role for lipids in retrotransposition.

Non-LTR retrotransposons are mobile genetic elements and play a major role in eukaryotic genome evolution and disease. Similar to retroviruses they encode a reverse transcriptase, but their genomic integration mechanism is fundamentally different, and they lack homologs of the retroviral nucleocapsid-forming protein Gag. Instead, their first open reading frames encode distinct multi-domain proteins (ORF1ps) presumed to package the retrotransposon-encoded RNA into ribonucleoprotein particles (RNPs). The mechanistic roles of ORF1ps are poorly understood, particularly of ORF1ps that appear to harbor an enzymatic function in the form of an SGNH-type lipolytic acetylesterase. We determined the crystal structures of the coiled coil and esterase domains of the ORF1p from the Danio rerio ZfL2-1 element. We demonstrate a dimerization of the coiled coil and a hydrolytic activity of the esterase. Furthermore, the esterase binds negatively charged phospholipids and liposomes, but not oligo-(A) RNA. Unexpectedly, the esterase can split into two dynamic half-domains, suited to engulf long fatty acid substrates extending from the active site. These properties indicate a role for lipids and membranes in non-LTR retrotransposition. We speculate that Gag-like membrane targeting properties of ORF1ps could play a role in RNP assembly and in membrane-dependent transport or localization processes.

Small RNA binding to the lateral surface of Hfq hexamers and structural rearrangements upon mRNA target recognition.

The bacterial Sm-like protein Hfq is a central player in the control of bacterial gene expression. Hfq forms complexes with small regulatory RNAs (sRNAs) that use complementary "seed" sequences to target specific mRNAs. Hfq forms hexameric rings, which preferably bind uridine-rich RNA 3' ends on their proximal surface and adenine-rich sequences on their distal surface. However, many reported properties of Hfq/sRNA complexes could not be explained by these RNA binding modes. Here, we use the RybB sRNA to identify the lateral surface of Hfq as a third, independent RNA binding surface. A systematic mutational analysis and competition experiments demonstrate that the lateral sites have a preference for and are sufficient to bind the sRNA "body," including the seed sequence. Furthermore, we detect significant structural rearrangements of the Hfq/sRNA complex upon mRNA target recognition that lead to a release of the seed sequence, or of the entire sRNA molecule in case of an unfavorable 3' end. Consequently, we propose a molecular model for the Hfq/sRNA complex, where the sRNA 3' end is anchored in the proximal site of Hfq, whereas the sRNA body, including the seed sequence, is bound by up to six of the lateral sites. In contrast to previously proposed arrangements, the presented model explains how Hfq can protect large parts of the sRNA body while still allowing a rapid recycling of sRNAs. Furthermore, our model suggests molecular mechanisms for the function of Hfq as an RNA chaperone and for the molecular events that are initiated upon mRNA target recognition.

The C-terminal alpha-alpha 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 alpha-alpha 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.

RNA binding by Hfq and ring-forming (L)Sm proteins: a trade-off between optimal sequence readout and RNA backbone conformation.

The eukaryotic Sm and the Sm-like (LSm) proteins form a large family that includes LSm proteins in archaea and the Hfq proteins in bacteria. Commonly referred to as the (L)Sm protein family, the various members play important roles in RNA processing, decay, and riboregulation. Particularly interesting from a structural point of view is their ability to assemble into doughnut-shaped rings, which allows them to bind preferentially the uridine-rich 3'-end of RNA oligonucleotides. With an emphasis on Hfq, this review compares the RNA-binding properties of the various (L)Sm rings that were recently co-crystallized with RNA substrates, and it discusses how these properties relate to physiological function.