UPF1 helicase orchestrates mutually exclusive interactions with the SMG6 endonuclease and UPF2.

Nonsense-mediated mRNA decay (NMD) is a conserved co-translational mRNA surveillance and turnover pathway across eukaryotes. NMD has a central role in degrading defective mRNAs and also regulates the stability of a significant portion of the transcriptome. The pathway is organized around UPF1, an RNA helicase that can interact with several NMD-specific factors. In human cells, degradation of the targeted mRNAs begins with a cleavage event that requires the recruitment of the SMG6 endonuclease to UPF1. Previous studies have identified functional links between SMG6 and UPF1, but the underlying molecular mechanisms have remained elusive. Here, we used mass spectrometry, structural biology and biochemical approaches to identify and characterize a conserved short linear motif in SMG6 that interacts with the cysteine/histidine-rich (CH) domain of UPF1. Unexpectedly, we found that the UPF1-SMG6 interaction is precluded when the UPF1 CH domain is engaged with another NMD factor, UPF2. Based on cryo-EM data, we propose that the formation of distinct SMG6-containing and UPF2-containing NMD complexes may be dictated by different conformational states connected to the RNA-binding status of UPF1. Our findings rationalize a key event in metazoan NMD and advance our understanding of mechanisms regulating activity and guiding substrate recognition by the SMG6 endonuclease.

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding.

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo-complex. We found that the Nse5/6 sub-complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross-linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross-linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non-productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

RNA degradation paths in a 12-subunit nuclear exosome complex.

The eukaryotic exosome is a conserved RNA-degrading complex that functions in RNA surveillance, turnover and processing. How the same machinery can either completely degrade or precisely trim RNA substrates has long remained unexplained. Here we report the crystal structures of a yeast nuclear exosome containing the 9-subunit core, the 3'-5' RNases Rrp44 and Rrp6, and the obligate Rrp6-binding partner Rrp47 in complex with different RNAs. The combined structural and biochemical data of this 12-subunit complex reveal how a single-stranded RNA can reach the Rrp44 or Rrp6 active sites directly or can bind Rrp6 and be threaded via the central channel towards the distal RNase Rrp44. When a bulky RNA is stalled at the entrance of the channel, Rrp6-Rrp47 swings open. The results suggest how the same molecular machine can coordinate processive degradation and partial trimming in an RNA-dependent manner by a concerted swinging mechanism of the two RNase subunits.

Architecture of the nuclease module of the yeast Ccr4-not complex: the Not1-Caf1-Ccr4 interaction.

Shortening eukaryotic poly(A) tails represses mRNA translation and induces mRNA turnover. The major cytoplasmic deadenylase, the Ccr4-Not complex, is a conserved multisubunit assembly. Ccr4-Not is organized around Not1, a large scaffold protein that recruits two 3'-5' exoribonucleases, Caf1 and Ccr4. We report structural studies showing that the N-terminal arm of yeast Not1 has a HEAT-repeat structure with domains related to the MIF4G fold. A MIF4G domain positioned centrally within the Not1 protein recognizes Caf1, which in turn binds the LRR domain of Ccr4 and tethers the Ccr4 nuclease domain. The interactions that form the nuclease core of the Ccr4-Not complex are evolutionarily conserved. Their specific disruption affects cell growth and mRNA deadenylation and decay in vivo in yeast. Thus, the N-terminal arm of Not1 forms an extended platform reminiscent of scaffolding proteins like eIF4G and CBP80, and places the two nucleases in a pivotal position within the Ccr4-Not complex.

Structure of a SMG8-SMG9 complex identifies a G-domain heterodimer in the NMD effector proteins.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA degradation pathway involved in surveillance and post-transcriptional regulation, and executed by the concerted action of several trans-acting factors. The SMG1 kinase is an essential NMD factor in metazoans and is associated with two recently identified and yet poorly characterized proteins, SMG8 and SMG9. We determined the 2.5 Å resolution crystal structure of a SMG8-SMG9 core complex from C. elegans We found that SMG8-SMG9 is a G-domain heterodimer with architectural similarities to the dynamin-like family of GTPases such as Atlastin and GBP1. The SMG8-SMG9 heterodimer forms in the absence of nucleotides, with interactions conserved from worms to humans. Nucleotide binding occurs at the G domain of SMG9 but not of SMG8. Fitting the GDP-bound SMG8-SMG9 structure in EM densities of the human SMG1-SMG8-SMG9 complex raises the possibility that the nucleotide site of SMG9 faces SMG1 and could impact the kinase conformation and/or regulation.

Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2.

Upf1 is a crucial factor in nonsense-mediated mRNA decay, the eukaryotic surveillance pathway that degrades mRNAs containing premature stop codons. The essential RNA-dependent ATPase activity of Upf1 is triggered by the formation of the surveillance complex with Upf2-Upf3. We report crystal structures of Upf1 in the presence and absence of the CH domain, captured in the transition state with ADP:AlF4⁻ and RNA. In isolation, Upf1 clamps onto the RNA, enclosing it in a channel formed by both the catalytic and regulatory domains. Upon binding to Upf2, the regulatory CH domain of Upf1 undergoes a large conformational change, causing the catalytic helicase domain to bind RNA less extensively and triggering its helicase activity. Formation of the surveillance complex thus modifies the RNA binding properties and the catalytic activity of Upf1, causing it to switch from an RNA-clamping mode to an RNA-unwinding mode.

The exosome-binding factors Rrp6 and Rrp47 form a composite surface for recruiting the Mtr4 helicase.

The exosome is a conserved multi-subunit ribonuclease complex that functions in 3' end processing, turnover and surveillance of nuclear and cytoplasmic RNAs. In the yeast nucleus, the 10-subunit core complex of the exosome (Exo-10) physically and functionally interacts with the Rrp6 exoribonuclease and its associated cofactor Rrp47, the helicase Mtr4 and Mpp6. Here, we show that binding of Mtr4 to Exo-10 in vitro is dependent upon both Rrp6 and Rrp47, whereas Mpp6 binds directly and independently of other cofactors. Crystallographic analyses reveal that the N-terminal domains of Rrp6 and Rrp47 form a highly intertwined structural unit. Rrp6 and Rrp47 synergize to create a composite and conserved surface groove that binds the N-terminus of Mtr4. Mutation of conserved residues within Rrp6 and Mtr4 at the structural interface disrupts their interaction and inhibits growth of strains expressing a C-terminal GFP fusion of Mtr4. These studies provide detailed structural insight into the interaction between the Rrp6-Rrp47 complex and Mtr4, revealing an important link between Mtr4 and the core exosome.

Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity.

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2-GLD-3, we report here the 2.5 Å resolution structure and biochemical characterization of a GLD-2-RNP-8 core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes.

HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment.

Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase-telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC-based DNA-protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere-binding protein 1 (HOT1). HOT1 directly and specifically binds double-stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase-dependent telomere elongation.

Crystal structure of the human eIF4AIII-CWC22 complex shows how a DEAD-box protein is inhibited by a MIF4G domain.

DEAD-box proteins are involved in all aspects of RNA processing. They bind RNA in an ATP-dependent manner and couple ATP hydrolysis to structural and compositional rearrangements of ribonucleoprotein particles. Conformational control is a major point of regulation for DEAD-box proteins to act on appropriate substrates and in a timely manner in vivo. Binding partners containing a middle domain of translation initiation factor 4G (MIF4G) are emerging as important regulators. Well-known examples are eIF4G and Gle1, which bind and activate the DEAD-box proteins eIF4A and Dbp5. Here, we report the mechanism of an inhibiting MIF4G domain. We determined the 2.0-Å resolution structure of the complex of human eIF4AIII and the MIF4G domain of the splicing factor Complexed With Cef1 (CWC22), an essential prerequisite for exon junction complex assembly by the splicing machinery. The CWC22 MIF4G domain binds both RecA domains of eIF4AIII. The mode of RecA2 recognition is similar to that observed in the activating complexes, yet is specific for eIF4AIII. The way the CWC22 MIF4G domain latches on the eIF4AIII RecA1 domain is markedly different from activating complexes. In the CWC22-eIF4AIII complex, the RNA-binding and ATP-binding motifs of the two RecA domains do not face each other, as would be required in the active state, but are in diametrically opposite positions. The binding mode of CWC22 to eIF4AIII reveals a facet of how MIF4G domains use their versatile structural frameworks to activate or inhibit DEAD-box proteins.

Crystal structure of the intraflagellar transport complex 25/27.

The cilium is an important organelle that is found on many eukaryotic cells, where it serves essential functions in motility, sensory reception and signalling. Intraflagellar transport (IFT) is a vital process for the formation and maintenance of cilia. We have determined the crystal structure of Chlamydomonas reinhardtii IFT25/27, an IFT sub-complex, at 2.6 Å resolution. IFT25 and IFT27 interact via a conserved interface that we verify biochemically using structure-guided mutagenesis. IFT27 displays the fold of Rab-like small guanosine triphosphate hydrolases (GTPases), binds GTP and GDP with micromolar affinity and has very low intrinsic GTPase activity, suggesting that it likely requires a GTPase-activating protein (GAP) for robust GTP turnover. A patch of conserved surface residues contributed by both IFT25 and IFT27 is found adjacent to the GTP-binding site and could mediate the binding to other IFT proteins as well as to a potential GAP. These results provide the first step towards a high-resolution structural understanding of the IFT complex.

Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1, Edc3 and RNA in mutually exclusive interactions.

Translational repression and deadenylation of eukaryotic mRNAs result either in the sequestration of the transcripts in a nontranslatable pool or in their degradation. Removal of the 5' cap structure is a crucial step that commits deadenylated mRNAs to 5'-to-3' degradation. Pat1, Edc3 and the DEAD-box protein Dhh1 are evolutionary conserved factors known to participate in both translational repression and decapping, but their interplay is currently unclear. We report the 2.8 Å resolution structure of yeast Dhh1 bound to the N-terminal domain of Pat1. The structure shows how Pat1 wraps around the C-terminal RecA domain of Dhh1, docking onto the Phe-Asp-Phe (FDF) binding site. The FDF-binding site of Dhh1 also recognizes Edc3, revealing why the binding of Pat1 and Edc3 on Dhh1 are mutually exclusive events. Using co-immunoprecipitation assays and structure-based mutants, we demonstrate that the mode of Dhh1-Pat1 recognition is conserved in humans. Pat1 and Edc3 also interfere and compete with the RNA-binding properties of Dhh1. Mapping the RNA-binding sites on Dhh1 with a crosslinking-mass spectrometry approach shows a large RNA-binding surface around the C-terminal RecA domain, including the FDF-binding pocket. The results suggest a model for how Dhh1-containing messenger ribonucleoprotein particles might be remodeled upon Pat1 and Edc3 binding.

Structural and biochemical studies of SLIP1-SLBP identify DBP5 and eIF3g as SLIP1-binding proteins.

In metazoans, replication-dependent histone mRNAs end in a stem-loop structure instead of the poly(A) tail characteristic of all other mature mRNAs. This specialized 3' end is bound by stem-loop binding protein (SLBP), a protein that participates in the nuclear export and translation of histone mRNAs. The translational activity of SLBP is mediated by interaction with SLIP1, a middle domain of initiation factor 4G (MIF4G)-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of zebrafish SLIP1 bound to the translation-activation domain of SLBP and identified the determinants of the recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. We confirmed the binding of SLIP1 to DBP5 and eIF3g by pull-down assays and determined the 3.25 Å resolution structure of SLIP1 bound to the DBP5 SBM. The SBM-binding and homodimerization residues of SLIP1 are conserved in the MIF4G domain of CBP80/20-dependent translation initiation factor (CTIF). The results suggest how the SLIP1 homodimer or a SLIP1-CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.

Structural analyses of the CRISPR protein Csc2 reveal the RNA-binding interface of the type I-D Cas7 family.

Upon pathogen invasion, bacteria and archaea activate an RNA-interference-like mechanism termed CRISPR (clustered regularly interspaced short palindromic repeats). A large family of Cas (CRISPR-associated) proteins mediates the different stages of this sophisticated immune response. Bioinformatic studies have classified the Cas proteins into families, according to their sequences and respective functions. These range from the insertion of the foreign genetic elements into the host genome to the activation of the interference machinery as well as target degradation upon attack. Cas7 family proteins are central to the type I and type III interference machineries as they constitute the backbone of the large interference complexes. Here we report the crystal structure of Thermofilum pendens Csc2, a Cas7 family protein of type I-D. We found that Csc2 forms a core RRM-like domain, flanked by three peripheral insertion domains: a lid domain, a Zinc-binding domain and a helical domain. Comparison with other Cas7 family proteins reveals a set of similar structural features both in the core and in the peripheral domains, despite the absence of significant sequence similarity. T. pendens Csc2 binds single-stranded RNA in vitro in a sequence-independent manner. Using a crosslinking - mass-spectrometry approach, we mapped the RNA-binding surface to a positively charged surface patch on T. pendens Csc2. Thus our analysis of the key structural and functional features of T. pendens Csc2 highlights recurring themes and evolutionary relationships in type I and type III Cas proteins.

Insights into the recruitment of the NMD machinery from the crystal structure of a core EJC-UPF3b complex.

In mammals, Up-frameshift proteins (UPFs) form a surveillance complex that interacts with the exon junction complex (EJC) to elicit nonsense-mediated mRNA decay (NMD). UPF3b is the component of the surveillance complex that bridges the interaction with the EJC. Here, we report the 3.4 A resolution crystal structure of a minimal UPF3b-EJC assembly, consisting of the interacting domains of five proteins (UPF3b, MAGO, Y14, eIF4AIII, and Barentsz) together with RNA and adenylyl-imidodiphosphate. Human UPF3b binds with the C-terminal domain stretched over a composite surface formed by eIF4AIII, MAGO, and Y14. Residues that affect NMD when mutated are found at the core interacting surfaces, whereas differences between UPF3b and UPF3a map at peripheral interacting residues. Comparison with the binding mode of the protein PYM underscores how a common molecular surface of MAGO and Y14 recognizes different proteins acting at different times in the same pathway. The binding mode to eIF4AIII identifies a surface hot spot that is used by different DEAD-box proteins to recruit their regulators.

Suramin inhibits SARS-CoV-2 nucleocapsid phosphoprotein genome packaging function.

The coronavirus disease 2019 (COVID-19) pandemic is fading, however its etiologic agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues posing - despite the availability of licensed vaccines - a global health threat, due to the potential emergence of vaccine-resistant SARS-CoV-2 variants. This makes the development of new drugs against COVID-19 a persistent urgency and sets as research priority the validation of novel therapeutic targets within the SARS-CoV-2 proteome. Among these, a promising one is the SARS-CoV-2 nucleocapsid (N) phosphoprotein, a major structural component of the virion with indispensable role in packaging the viral genome into a ribonucleoprotein (RNP) complex, which also contributes to SARS-CoV-2 innate immune evasion by inhibiting the host cell type-I interferon (IFN-I) response. By combining miniaturized differential scanning fluorimetry with microscale thermophoresis, we found that the 100-year-old drug Suramin interacts with SARS-CoV-2 N-terminal domain (NTD) and C-terminal domain (CTD), thereby inhibiting their single-stranded RNA (ssRNA) binding function with low-micromolar Kd and IC50 values. Molecular docking suggests that Suramin interacts with basic NTD cleft and CTD dimer interface groove, highlighting three potentially druggable ssRNA binding sites. Electron microscopy shows that Suramin inhibits the formation in vitro of RNP complex-like condensates by SARS-CoV-2 N with a synthetic ssRNA. In a dose-dependent manner, Suramin also reduced SARS-CoV-2-induced cytopathic effect on Vero E6 and Calu-3 cells, partially reverting the SARS-CoV-2 N-inhibited IFN-I production in 293T cells. Our findings indicate that Suramin inhibits SARS-CoV-2 replication by hampering viral genome packaging, thereby representing a starting model for design of new COVID-19 antivirals.

The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity.

During mRNA localization, RNA-binding proteins interact with specific structured mRNA localization motifs. Although several such motifs have been identified, we have limited structural information on how these interact with RNA-binding proteins. Staufen proteins bind structured mRNA motifs through dsRNA-binding domains (dsRBD) and are involved in mRNA localization in Drosophila and mammals. We solved the structure of two dsRBDs of human Staufen1 in complex with a physiological dsRNA sequence. We identified interactions between the dsRBDs and the RNA sugar-phosphate backbone and direct contacts of conserved Staufen residues to RNA bases. Mutating residues mediating nonspecific backbone interactions only affected Staufen function in Drosophila when in vitro binding was severely reduced. Conversely, residues involved in base-directed interactions were required in vivo even when they minimally affected in vitro binding. Our work revealed that Staufen can read sequence features in the minor groove of dsRNA and suggests that these influence target selection in vivo.

The bicoid mRNA localization factor Exuperantia is an RNA-binding pseudonuclease.

Anterior patterning in Drosophila is mediated by the localization of bicoid (bcd) mRNA at the anterior pole of the oocyte. Exuperantia (Exu) is a putative exonuclease (EXO) associated with bcd and required for its localization. We present the crystal structure of Exu, which reveals a dimeric assembly with each monomer consisting of a 3'-5' EXO-like domain and a sterile alpha motif (SAM)-like domain. The catalytic site is degenerate and inactive. Instead, the EXO-like domain mediates dimerization and RNA binding. We show that Exu binds RNA directly in vitro, that the SAM-like domain is required for RNA binding activity and that Exu binds a structured element present in the bcd 3' untranslated region with high affinity. Through structure-guided mutagenesis, we show that Exu dimerization is essential for bcd localization. Our data demonstrate that Exu is a noncanonical RNA-binding protein with EXO-SAM-like domain architecture that interacts with its target RNA as a homodimer.

Structure of Rab11-FIP3-Rabin8 reveals simultaneous binding of FIP3 and Rabin8 effectors to Rab11.

The small GTPase Rab11 and its effectors FIP3 and Rabin8 are essential to membrane-trafficking pathways required for cytokinesis and ciliogenesis. Although effector binding is generally assumed to be sequential and mutually exclusive, we show that Rab11 can simultaneously bind FIP3 and Rabin8. We determined crystal structures of human Rab11-GMPPNP-Rabin8 and Rab11-GMPPNP-FIP3-Rabin8. The structures reveal that the C-terminal domain of Rabin8 adopts a previously undescribed fold that interacts with Rab11 at an unusual effector-binding site neighboring the canonical FIP3-binding site. We show that Rab11-GMPPNP-FIP3-Rabin8 is more stable than Rab11-GMPPNP-Rabin8, owing to direct interaction between Rabin8 and FIP3 within the dual effector-bound complex. The data allow us to propose a model for how membrane-targeting complexes assemble at the trans-Golgi network and recycling endosomes, through multiple weak interactions that create high-avidity complexes.

Deep transcriptome-sequencing and proteome analysis of the hydrothermal vent annelid Alvinella pompejana identifies the CvP-bias as a robust measure of eukaryotic thermostability.

BACKGROUND: Alvinella pompejana is an annelid worm that inhabits deep-sea hydrothermal vent sites in the Pacific Ocean. Living at a depth of approximately 2500 meters, these worms experience extreme environmental conditions, including high temperature and pressure as well as high levels of sulfide and heavy metals. A. pompejana is one of the most thermotolerant metazoans, making this animal a subject of great interest for studies of eukaryotic thermoadaptation. RESULTS: In order to complement existing EST resources we performed deep sequencing of the A. pompejana transcriptome. We identified several thousand novel protein-coding transcripts, nearly doubling the sequence data for this annelid. We then performed an extensive survey of previously established prokaryotic thermoadaptation measures to search for global signals of thermoadaptation in A. pompejana in comparison with mesophilic eukaryotes. In an orthologous set of 457 proteins, we found that the best indicator of thermoadaptation was the difference in frequency of charged versus polar residues (CvP-bias), which was highest in A. pompejana. CvP-bias robustly distinguished prokaryotic thermophiles from prokaryotic mesophiles, as well as the thermophilic fungus Chaetomium thermophilum from mesophilic eukaryotes. Experimental values for thermophilic proteins supported higher CvP-bias as a measure of thermal stability when compared to their mesophilic orthologs. Proteome-wide mean CvP-bias also correlated with the body temperatures of homeothermic birds and mammals. CONCLUSIONS: Our work extends the transcriptome resources for A. pompejana and identifies the CvP-bias as a robust and widely applicable measure of eukaryotic thermoadaptation.