Structural basis of transposon end recognition explains central features of Tn7 transposition systems.

Tn7 is a bacterial transposon with relatives containing element-encoded CRISPR-Cas systems mediating RNA-guided transposon insertion. Here, we present the 2.7 Å cryoelectron microscopy structure of prototypic Tn7 transposase TnsB interacting with the transposon end DNA. When TnsB interacts across repeating binding sites, it adopts a beads-on-a-string architecture, where the DNA-binding and catalytic domains are arranged in a tiled and intertwined fashion. The DNA-binding domains form few base-specific contacts leading to a binding preference that requires multiple weakly conserved sites at the appropriate spacing to achieve DNA sequence specificity. TnsB binding imparts differences in the global structure of the protein-bound DNA ends dictated by the spacing or overlap of binding sites explaining functional differences in the left and right ends of the element. We propose a model of the strand-transfer complex in which the terminal TnsB molecule is rearranged so that its catalytic domain is in a position conducive to transposition.

A heterotypic assembly mechanism regulates CHIP E3 ligase activity.

CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.

Structure-functional characterization of Lactococcus AbiA phage defense system.

Bacterial reverse transcriptases (RTs) are a large and diverse enzyme family. AbiA, AbiK and Abi-P2 are abortive infection system (Abi) RTs that mediate defense against bacteriophages. What sets Abi RTs apart from other RT enzymes is their ability to synthesize long DNA products of random sequences in a template- and primer-independent manner. Structures of AbiK and Abi-P2 representatives have recently been determined, but there are no structural data available for AbiA. Here, we report the crystal structure of Lactococcus AbiA polymerase in complex with a single-stranded polymerization product. AbiA comprises three domains: an RT-like domain, a helical domain that is typical for Abi polymerases, and a higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain that is common for many antiviral proteins. AbiA forms a dimer that distinguishes it from AbiK and Abi-P2, which form trimers/hexamers. We show the DNA polymerase activity of AbiA in an in vitro assay and demonstrate that it requires the presence of the HEPN domain which is enzymatically inactive. We validate our biochemical and structural results in vivo through bacteriophage infection assays. Finally, our in vivo results suggest that AbiA-mediated phage defense may not rely on AbiA-mediated cell death.

Despite the odds: formation of the SARS-CoV-2 methylation complex.

Coronaviruses modify their single-stranded RNA genome with a methylated cap during replication to mimic the eukaryotic mRNAs. The capping process is initiated by several nonstructural proteins (nsp) encoded in the viral genome. The methylation is performed by two methyltransferases, nsp14 and nsp16, while nsp10 acts as a co-factor to both. Additionally, nsp14 carries an exonuclease domain which operates in the proofreading system during RNA replication of the viral genome. Both nsp14 and nsp16 were reported to independently bind nsp10, but the available structural information suggests that the concomitant interaction between these three proteins would be impossible due to steric clashes. Here, we show that nsp14, nsp10, and nsp16 can form a heterotrimer complex upon significant allosteric change. This interaction is expected to encourage the formation of mature capped viral mRNA, modulating nsp14's exonuclease activity, and protecting the viral RNA. Our findings show that nsp14 is amenable to allosteric regulation and may serve as a novel target for therapeutic approaches.

Cryo-EM structure of rotavirus B NSP2 reveals its unique tertiary architecture.

Rotavirus (RV) NSP2 is a multifunctional RNA chaperone that exhibits numerous activities that are essential for replication and viral genome packaging. We performed an in silico analysis that highlighted a distant relationship of NSP2 from rotavirus B (RVB) to proteins from other human RVs. We solved a cryo-electron microscopy structure of RVB NSP2 that shows structural differences with corresponding proteins from other human RVs. Based on the structure, we identified amino acid residues that are involved in RNA interactions. Anisotropy titration experiments showed that these residues are important for nucleic acid binding. We also identified structural motifs that are conserved in all RV species. Collectively, our data complete the structural characterization of rotaviral NSP2 protein and demonstrate its structural diversity among RV species.IMPORTANCERotavirus B (RVB), also known as adult diarrhea rotavirus, has caused epidemics of severe diarrhea in China, India, and Bangladesh. Thousands of people are infected in a single RVB epidemic. However, information on this group of rotaviruses remains limited. As NSP2 is an essential protein in the viral life cycle, including its role in the formation of replication factories, it may be a target for future antiviral strategy against viruses with similar mechanisms.

Structures of annexin A2-PS DNA complexes show dominance of hydrophobic interactions in phosphorothioate binding.

The introduction of phosphorothioate (PS) linkages to the backbone of therapeutic nucleic acids substantially increases their stability and potency. It also affects their interactions with cellular proteins, but the molecular mechanisms that underlie this effect are poorly understood. Here, we report structural and biochemical studies of interactions between annexin A2, a protein that does not possess any known canonical DNA binding domains, and phosphorothioate-modified antisense oligonucleotides. We show that a unique mode of hydrophobic interactions between a sulfur atom of the phosphorothioate group and lysine and arginine residues account for the enhanced affinity of modified nucleic acid for the protein. Our results demonstrate that this mechanism of interaction is observed not only for nucleic acid-binding proteins but can also account for the association of PS oligonucleotides with other proteins. Using the anomalous diffraction of sulfur, we showed that preference for phosphorothioate stereoisomers is determined by the hydrophobic environment around the PS linkage that comes not only from protein but also from additional structural features within the ASO such as 5-Me groups on cytosine nucleobases.

The Pet127 protein is a mitochondrial 5'-to-3' exoribonuclease from the PD-(D/E)XK superfamily involved in RNA maturation and intron degradation in yeasts.

Pet127 is a mitochondrial protein found in multiple eukaryotic lineages, but absent from several taxa, including plants and animals. Distant homology suggests that it belongs to the divergent PD-(D/E)XK superfamily which includes various nucleases and related proteins. Earlier yeast genetics experiments suggest that it plays a nonessential role in RNA degradation and 5' end processing. Our phylogenetic analysis suggests that it is a primordial eukaryotic invention that was retained in diverse groups, and independently lost several times in the evolution of other organisms. We demonstrate for the first time that the fungal Pet127 protein in vitro is a processive 5'-to-3' exoribonuclease capable of digesting various substrates in a sequence nonspecific manner. Mutations in conserved residues essential in the PD-(D/E)XK superfamily active site abolish the activity of Pet127. Deletion of the PET127 gene in the pathogenic yeast Candida albicans results in a moderate increase in the steady-state levels of several transcripts and in accumulation of unspliced precursors and intronic sequences of three introns. Mutations in the active site residues result in a phenotype identical to that of the deletant, confirming that the exoribonuclease activity is related to the physiological role of the Pet127 protein. Pet127 activity is, however, not essential for maintaining the mitochondrial respiratory activity in C. albicans.

Mechanism of protein-primed template-independent DNA synthesis by Abi polymerases.

Abortive infection (Abi) is a bacterial antiphage defense strategy involving suicide of the infected cell. Some Abi pathways involve polymerases that are related to reverse transcriptases. They are unique in the way they combine the ability to synthesize DNA in a template-independent manner with protein priming. Here, we report crystal and cryo-electron microscopy structures of two Abi polymerases: AbiK and Abi-P2. Both proteins adopt a bilobal structure with an RT-like domain that comprises palm and fingers subdomains and a unique helical domain. AbiK and Abi-P2 adopt a hexameric and trimeric configuration, respectively, which is unprecedented for reverse transcriptases. Biochemical experiments showed that the formation of these oligomers is required for the DNA polymerization activity. The structure of the AbiK-DNA covalent adduct visualized interactions between the 3' end of DNA and the active site and covalent attachment of the 5' end of DNA to a tyrosine residue used for protein priming. Our data reveal a structural basis of the mechanism of highly unusual template-independent protein-priming polymerases.

cGAS surveillance of micronuclei links genome instability to innate immunity.

DNA is strictly compartmentalized within the nucleus to prevent autoimmunity; despite this, cyclic GMP-AMP synthase (cGAS), a cytosolic sensor of double-stranded DNA, is activated in autoinflammatory disorders and by DNA damage. Precisely how cellular DNA gains access to the cytoplasm remains to be determined. Here, we report that cGAS localizes to micronuclei arising from genome instability in a mouse model of monogenic autoinflammation, after exogenous DNA damage and spontaneously in human cancer cells. Such micronuclei occur after mis-segregation of DNA during cell division and consist of chromatin surrounded by its own nuclear membrane. Breakdown of the micronuclear envelope, a process associated with chromothripsis, leads to rapid accumulation of cGAS, providing a mechanism by which self-DNA becomes exposed to the cytosol. cGAS is activated by chromatin, and consistent with a mitotic origin, micronuclei formation and the proinflammatory response following DNA damage are cell-cycle dependent. By combining live-cell laser microdissection with single cell transcriptomics, we establish that interferon-stimulated gene expression is induced in micronucleated cells. We therefore conclude that micronuclei represent an important source of immunostimulatory DNA. As micronuclei formed from lagging chromosomes also activate this pathway, recognition of micronuclei by cGAS may act as a cell-intrinsic immune surveillance mechanism that detects a range of neoplasia-inducing processes.

Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase.

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

Structural and biochemical insights to the role of the CCR4-NOT complex and DDX6 ATPase in microRNA repression.

MicroRNAs (miRNAs) control gene expression by regulating mRNA translation and stability. The CCR4-NOT complex is a key effector of miRNA function acting downstream of GW182/TNRC6 proteins. We show that miRNA-mediated repression requires the central region of CNOT1, the scaffold protein of CCR4-NOT. A CNOT1 domain interacts with CNOT9, which in turn interacts with the silencing domain of TNRC6 in a tryptophan motif-dependent manner. These interactions are direct, as shown by the structure of a CNOT9-CNOT1 complex with bound tryptophan. Another domain of CNOT1 with an MIF4G fold recruits the DEAD-box ATPase DDX6, a known translational inhibitor. Structural and biochemical approaches revealed that CNOT1 modulates the conformation of DDX6 and stimulates ATPase activity. Structure-based mutations showed that the CNOT1 MIF4G-DDX6 interaction is important for miRNA-mediated repression. These findings provide insights into the repressive steps downstream of the GW182/TNRC6 proteins and the role of the CCR4-NOT complex in posttranscriptional regulation in general.

The expression of Rpb10, a small subunit common to RNA polymerases, is modulated by the R3H domain-containing Rbs1 protein and the Upf1 helicase.

The biogenesis of eukaryotic RNA polymerases is poorly understood. The present study used a combination of genetic and molecular approaches to explore the assembly of RNA polymerase III (Pol III) in yeast. We identified a regulatory link between Rbs1, a Pol III assembly factor, and Rpb10, a small subunit that is common to three RNA polymerases. Overexpression of Rbs1 increased the abundance of both RPB10 mRNA and the Rpb10 protein, which correlated with suppression of Pol III assembly defects. Rbs1 is a poly(A)mRNA-binding protein and mutational analysis identified R3H domain to be required for mRNA interactions and genetic enhancement of Pol III biogenesis. Rbs1 also binds to Upf1 protein, a key component in nonsense-mediated mRNA decay (NMD) and levels of RPB10 mRNA were increased in a upf1Δ strain. Genome-wide RNA binding by Rbs1 was characterized by UV cross-linking based approach. We demonstrated that Rbs1 directly binds to the 3' untranslated regions (3'UTRs) of many mRNAs including transcripts encoding Pol III subunits, Rpb10 and Rpc19. We propose that Rbs1 functions by opposing mRNA degradation, at least in part mediated by NMD pathway. Orthologues of Rbs1 protein are present in other eukaryotes, including humans, suggesting that this is a conserved regulatory mechanism.

Structure and mechanism of CutA, RNA nucleotidyl transferase with an unusual preference for cytosine.

Template-independent terminal ribonucleotide transferases (TENTs) catalyze the addition of nucleotide monophosphates to the 3'-end of RNA molecules regulating their fate. TENTs include poly(U) polymerases (PUPs) with a subgroup of 3' CUCU-tagging enzymes, such as CutA in Aspergillus nidulans. CutA preferentially incorporates cytosines, processively polymerizes only adenosines and does not incorporate or extend guanosines. The basis of this peculiar specificity remains to be established. Here, we describe crystal structures of the catalytic core of CutA in complex with an incoming non-hydrolyzable CTP analog and an RNA with three adenosines, along with biochemical characterization of the enzyme. The binding of GTP or a primer with terminal guanosine is predicted to induce clashes between 2-NH2 of the guanine and protein, which would explain why CutA is unable to use these ligands as substrates. Processive adenosine polymerization likely results from the preferential binding of a primer ending with at least two adenosines. Intriguingly, we found that the affinities of CutA for the CTP and UTP are very similar and the structures did not reveal any apparent elements for specific NTP binding. Thus, the properties of CutA likely result from an interplay between several factors, which may include a conformational dynamic process of NTP recognition.

Human REXO2 controls short mitochondrial RNAs generated by mtRNA processing and decay machinery to prevent accumulation of double-stranded RNA.

RNA decay is a key element of mitochondrial RNA metabolism. To date, the only well-documented machinery that plays a role in mtRNA decay in humans is the complex of polynucleotide phosphorylase (PNPase) and SUV3 helicase, forming the degradosome. REXO2, a homolog of prokaryotic oligoribonucleases present in humans both in mitochondria and the cytoplasm, was earlier shown to be crucial for maintaining mitochondrial homeostasis, but its function in mitochondria has not been fully elucidated. In the present study, we created a cellular model that enables the clear dissection of mitochondrial and non-mitochondrial functions of human REXO2. We identified a novel mitochondrial short RNA, referred to as ncH2, that massively accumulated upon REXO2 silencing. ncH2 degradation occurred independently of the mitochondrial degradosome, strongly supporting the hypothesis that ncH2 is a primary substrate of REXO2. We also investigated the global impact of REXO2 depletion on mtRNA, revealing the importance of the protein for maintaining low steady-state levels of mitochondrial antisense transcripts and double-stranded RNA. Our detailed biochemical and structural studies provide evidence of sequence specificity of the REXO2 oligoribonuclease. We postulate that REXO2 plays dual roles in human mitochondria, 'scavenging' nanoRNAs that are produced by the degradosome and clearing short RNAs that are generated by RNA processing.

Recognition and processing of branched DNA substrates by Slx1-Slx4 nuclease.

Structure-selective endonucleases cleave branched DNA substrates. Slx1 is unique among structure-selective nucleases because it can cleave all branched DNA structures at multiple sites near the branch point. The mechanism behind this broad range of activity is unknown. The present study structurally and biochemically investigated fungal Slx1 to define a new protein interface that binds the non-cleaved arm of branched DNAs. The DNA arm bound at this new site was positioned at a sharp angle relative to the arm that was modeled to interact with the active site, implying that Slx1 uses DNA bending to localize the branch point as a flexible discontinuity in DNA. DNA binding at the new interface promoted a disorder-order transition in a region of the protein that was located in the vicinity of the active site, potentially participating in its formation. This appears to be a safety mechanism that ensures that DNA cleavage occurs only when the new interface is occupied by the non-cleaved DNA arm. Models of Slx1 that interacted with various branched DNA substrates were prepared. These models explain the way in which Slx1 cuts DNA toward the 3' end away from the branch point and elucidate the unique ability of Slx1 to cleave various DNA structures.

Origins of the Increased Affinity of Phosphorothioate-Modified Therapeutic Nucleic Acids for Proteins.

The phosphorothioate backbone modification (PS) is one of the most widely used chemical modifications for enhancing the drug-like properties of nucleic acid-based drugs, including antisense oligonucleotides (ASOs). PS-modified nucleic acid therapeutics show improved metabolic stability from nuclease-mediated degradation and exhibit enhanced interactions with plasma, cell-surface, and intracellular proteins, which facilitates their tissue distribution and cellular uptake in animals. However, little is known about the structural basis of the interactions of PS nucleic acids with proteins. Here, we report a crystal structure of the DNA-binding domain of a model ASO-binding protein PC4, in complex with a full PS 2'-OMe DNA gapmer ASO. To our knowledge this is the first structure of a complex between a protein and fully PS nucleic acid. Each PC4 dimer comprises two DNA-binding interfaces. In the structure one interface binds the 5'-terminal 2'-OMe PS flank of the ASO, while the other interface binds the regular PS DNA central part in the opposite polarity. As a result, the ASO forms a hairpin-like structure. ASO binding also induces the formation of a dimer of dimers of PC4, which is stabilized by base pairing between homologous regions of the ASOs bound by each dimer of PC4. The protein interacts with the PS nucleic acid through a network of electrostatic and hydrophobic interactions, which provides insights into the origins for the enhanced affinity of PS for proteins. The importance of these contacts was further confirmed in a NanoBRET binding assay using a Nano luciferase tagged PC4 acting as the BRET donor, to a fluorescently conjugated ASO acting as the BRET acceptor. Overall, our results provide insights into the molecular forces that govern the interactions of PS ASOs with cellular proteins and provide a potential model for how these interactions can template protein-protein interactions causative of cellular toxicity.

Evolutionary History and Activity of RNase H1-Like Proteins in Arabidopsis thaliana.

RNase H1 is an endonuclease specific toward the RNA strand of RNA:DNA hybrids. Members of this protein family are present in most living organisms and are essential for removing RNA that base pairs with DNA. It prevents detrimental effects of RNA:DNA hybrids and is involved in several biological processes. Arabidopsis thaliana has been previously shown to contain three genes encoding RNase H1 proteins that localize to three distinct cellular compartments. We show that these genes originate from two gene duplication events. One occurred in the common ancestor of dicots and produced nuclear and organellar RNase H1 paralogs. Second duplication occurred in the common ancestor of Brassicaceae and produced mitochondrial- and plastid-localized proteins. These proteins have the canonical RNase H1 activity, which requires at least four ribonucleotides for endonucleolytic digestion. Analysis of mutants in the RNase H1 genes revealed that the nuclear RNH1A and mitochondrial RNH1B are dispensable for development under normal growth conditions. However, the presence of at least one organellar RNase H1 (RNH1B or RNH1C) is required for embryonic development. The plastid-localized RNH1C affects plastid DNA copy number and sensitivity to replicative stress. Our results present the evolutionary history of RNH1 proteins in A. thaliana, demonstrate their canonical RNase H1 activity and indicate their role in early embryonic development.

Disulfide bridge cross-linking between protein and the RNA backbone as a tool to study RNase H1.

The chemical cross-linking of complexes of proteins with nucleic acids is often used in structural and mechanistic studies of these oftentimes unstable and transient complexes. To date, no method has been reported for the thiol-based conjugation of proteins with an RNA backbone, mainly because of instability of the modified ribonucleic acid that is functionalized at the phosphodiester and its rapid hydrolysis. Here, we report the site-specific synthesis of stable RNA oligonucleotides with a thiol-bearing linker that was attached to the phosphodiester backbone, where the ribonucleotide at the cross-linking site was either replaced with 2'-deoxy- or 2'-fluororibonucleotide. The utility of this approach was validated in cross-linking tests with RNase H1, a model protein for RNA/DNA binding and key effector in DNA-like antisense drug therapy. Furthermore, scale-up cross-linking and purification of the complexes confirmed that the method is useful for obtaining preparations of protein-RNA/DNA complexes with purity and stability that are suitable for further biochemical and structural studies. The present approach broadens the repertoire of disulfide-based cross-linking strategies and is a novel tool for the stabilization of protein-RNA complexes in which the interaction occurs via the RNA backbone. This methodology may be broadly applicable to studies of otherwise unstable or transient complexes of proteins with RNA and RNA/DNA.

Mechanism of polypurine tract primer generation by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.