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.

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.

Cellular Traffic Jam and Disease Due to Mutations in SRP54.

In this issue of Structure, Juaire et al. use X-ray crystallography, biophysical tools, and cell-based assays to investigate disease-associated variants of the SRP54 GTPase and to demonstrate that defects in SRP-mediated protein secretion can explain phenotypes of severe neutropenia with Shwachman-Diamond-syndrome-like symptoms.

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.

Human LINE-1 retrotransposition requires a metastable coiled coil and a positively charged N-terminus in L1ORF1p.

LINE-1 (L1) is an autonomous retrotransposon, which acted throughout mammalian evolution and keeps contributing to human genotypic diversity, genetic disease and cancer. L1 encodes two essential proteins: L1ORF1p, a unique RNA-binding protein, and L1ORF2p, an endonuclease and reverse transcriptase. L1ORF1p contains an essential, but rapidly evolving N-terminal portion, homo-trimerizes via a coiled coil and packages L1RNA into large assemblies. Here, we determined crystal structures of the entire coiled coil domain of human L1ORF1p. We show that retrotransposition requires a non-ideal and metastable coiled coil structure, and a strongly basic L1ORF1p amino terminus. Human L1ORF1p therefore emerges as a highly calibrated molecular machine, sensitive to mutation but functional in different hosts. Our analysis rationalizes the locally rapid L1ORF1p sequence evolution and reveals striking mechanistic parallels to coiled coil-containing membrane fusion proteins. It also suggests how trimeric L1ORF1p could form larger meshworks and indicates critical novel steps in L1 retrotransposition.

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.

Mobilization of LINE-1 retrotransposons is restricted by Tex19.1 in mouse embryonic stem cells.

Mobilization of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution, but these mutagenic events can also cause genetic disorders. In humans, retrotransposon mobilization is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilize in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilization. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, loss of Tex19.1 increases L1-ORF1p levels and L1 mobilization in pluripotent mouse embryonic stem cells, implying that Tex19.1 prevents de novo retrotransposition in the pluripotent phase of the germline cycle. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in mammals.

Mille viae in eukaryotic mRNA decapping.

Cellular mRNA levels are regulated via rates of transcription and decay. Since the removal of the mRNA 5'-cap by the decapping enzyme DCP2 is generally an irreversible step towards decay, it requires regulation. Control of DCP2 activity is likely effected by two interdependent means: by conformational control of the DCP2-DCP1 complex, and by assembly control of the decapping network, an array of mutually interacting effector proteins. Here, we compare three recent and conformationally distinct crystal structures of the DCP2-DCP1 decapping complex in the presence of substrate analogs and decapping enhancers and we discuss alternative substrate recognition modes for the catalytic domain of DCP2. Together with structure-based insight into decapping network assembly, we propose that DCP2-mediated decapping follows more than one path.

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.

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.

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.

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.

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.

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.

An asymmetric PAN3 dimer recruits a single PAN2 exonuclease to mediate mRNA deadenylation and decay.

The PAN2-PAN3 complex functions in general and microRNA-mediated mRNA deadenylation. However, mechanistic insight into PAN2 and its complex with the asymmetric PAN3 dimer is lacking. Here, we describe crystal structures that show that Neurospora crassa PAN2 comprises two independent structural units: a C-terminal catalytic unit and an N-terminal assembly unit that engages in a bipartite interaction with PAN3 dimers. The catalytic unit contains the exonuclease domain in an intimate complex with a potentially modulatory ubiquitin-protease-like domain. The assembly unit contains a WD40 propeller connected to an adaptable linker. The propeller contacts the PAN3 C-terminal domain, whereas the linker reinforces the asymmetry of the PAN3 dimer and prevents the recruitment of a second PAN2 molecule. Functional data indicate an essential role for PAN3 in coordinating PAN2-mediated deadenylation with subsequent steps in mRNA decay, which lead to complete mRNA degradation.

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.

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.