Chaperoning SNARE Folding and Assembly.

SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.

A Small Helical Bundle Prepares Primer Synthesis by Binding Two Nucleotides that Enhance Sequence-Specific Recognition of the DNA Template.

Primases have a fundamental role in DNA replication. They synthesize a primer that is then extended by DNA polymerases. Archaeoeukaryotic primases require for synthesis a catalytic and an accessory domain, the exact contribution of the latter being unresolved. For the pRN1 archaeal primase, this domain is a 115-amino acid helix bundle domain (HBD). Our structural investigations of this small HBD by liquid- and solid-state nuclear magnetic resonance (NMR) revealed that only the HBD binds the DNA template. DNA binding becomes sequence-specific after a major allosteric change in the HBD, triggered by the binding of two nucleotide triphosphates. The spatial proximity of the two nucleotides and the DNA template in the quaternary structure of the HBD strongly suggests that this small domain brings together the substrates to prepare the first catalytic step of primer synthesis. This efficient mechanism is likely general for all archaeoeukaryotic primases.

Lesion Bypass and the Reactivation of Stalled Replication Forks.

Accurate transmission of the genetic information requires complete duplication of the chromosomal DNA each cell division cycle. However, the idea that replication forks would form at origins of DNA replication and proceed without impairment to copy the chromosomes has proven naive. It is now clear that replication forks stall frequently as a result of encounters between the replication machinery and template damage, slow-moving or paused transcription complexes, unrelieved positive superhelical tension, covalent protein-DNA complexes, and as a result of cellular stress responses. These stalled forks are a major source of genome instability. The cell has developed many strategies for ensuring that these obstructions to DNA replication do not result in loss of genetic information, including DNA damage tolerance mechanisms such as lesion skipping, whereby the replisome jumps the lesion and continues downstream; template switching both behind template damage and at the stalled fork; and the error-prone pathway of translesion synthesis.

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.

Despite the numerous sequencing methods available, the diversity in RNA size and chemical modification makes it difficult to capture all RNAs in a cell. We developed a method that combines quasi-random priming with template switching to construct sequencing libraries from RNA molecules of any length and with any type of 3' modifications, allowing for the sequencing of virtually all RNA species. Our ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to identify and quantify all classes of coding and non-coding RNAs. With LIDAR, we comprehensively characterized the transcriptomes of mouse embryonic stem cells, neural progenitor cells, mouse tissues, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared with traditional ligation-dependent sequencing methods and uncovered tDRs with blocked 3' ends that had previously escaped detection. Therefore, LIDAR can capture all RNAs in a sample and uncover RNA species with potential regulatory functions.

The SARS-CoV-2 subgenome landscape and its novel regulatory features.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is currently a global pandemic. CoVs are known to generate negative subgenomes (subgenomic RNAs [sgRNAs]) through transcription-regulating sequence (TRS)-dependent template switching, but the global dynamic landscapes of coronaviral subgenomes and regulatory rules remain unclear. Here, using next-generation sequencing (NGS) short-read and Nanopore long-read poly(A) RNA sequencing in two cell types at multiple time points after infection with SARS-CoV-2, we identified hundreds of template switches and constructed the dynamic landscapes of SARS-CoV-2 subgenomes. Interestingly, template switching could occur in a bidirectional manner, with diverse SARS-CoV-2 subgenomes generated from successive template-switching events. The majority of template switches result from RNA-RNA interactions, including seed and compensatory modes, with terminal pairing status as a key determinant. Two TRS-independent template switch modes are also responsible for subgenome biogenesis. Our findings reveal the subgenome landscape of SARS-CoV-2 and its regulatory features, providing a molecular basis for understanding subgenome biogenesis and developing novel anti-viral strategies.

Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses.

Viral RNA-dependent RNA polymerases (RdRps) are a target for broad-spectrum antiviral therapeutic agents. Recently, we demonstrated that incorporation of the T-1106 triphosphate, a pyrazine-carboxamide ribonucleotide, into nascent RNA increases pausing and backtracking by the poliovirus RdRp. Here, by monitoring enterovirus A-71 RdRp dynamics during RNA synthesis using magnetic tweezers, we identify the "backtracked" state as an intermediate used by the RdRp for copy-back RNA synthesis and homologous recombination. Cell-based assays and RNA sequencing (RNA-seq) experiments further demonstrate that the pyrazine-carboxamide ribonucleotide stimulates these processes during infection. These results suggest that pyrazine-carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.

Mitotic CDK Promotes Replisome Disassembly, Fork Breakage, and Complex DNA Rearrangements.

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.

Tinker, tailor, antiviral: RNA virus inhibition by induced recombination.

Nucleotide analogs can help to combat RNA virus growth by stalling the viral RNA polymerase or by introducing lethal mutations into the viral genome. Janissen and Woodman et al. have used single-molecule, sequencing, and virological methods to reveal that antiviral T-1106 provides a third mechanism of counterattack: inducing recombination.

Quantification of tRNA m1A modification by templated-ligation qPCR.

N1-methyladenosine (m1A) is a widespread modification in all eukaryotic, many archaeal, and some bacterial tRNAs. m1A is generally located in the T loop of cytosolic tRNA and between the acceptor and D stems of mitochondrial tRNAs; it is involved in the tertiary interaction that stabilizes tRNA. Human tRNA m1A levels are dynamically regulated that fine-tune translation and can also serve as biomarkers for infectious disease. Although many methods have been used to measure m1A, a PCR method to assess m1A levels quantitatively in specific tRNAs has been lacking. Here we develop a templated-ligation followed by a qPCR method (TL-qPCR) that measures m1A levels in target tRNAs. Our method uses the SplintR ligase that efficiently ligates two tRNA complementary DNA oligonucleotides using tRNA as the template, followed by qPCR using the ligation product as the template. m1A interferes with the ligation in specific ways, allowing for the quantitative assessment of m1A levels using subnanogram amounts of total RNA. We identify the features of specificity and quantitation for m1A-modified model RNAs and apply these to total RNA samples from human cells. Our method enables easy access to study the dynamics and function of this pervasive tRNA modification.

Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop.

Most HIV-1-specific neutralizing antibodies isolated to date exhibit unusual characteristics that complicate their elicitation. Neutralizing antibodies that target the V1V2 apex of the HIV-1 envelope (Env) trimer feature unusually long protruding loops, which enable them to penetrate the HIV-1 glycan shield. As antibodies with loops of requisite length are created through uncommon recombination events, an alternative mode of apex binding has been sought. Here, we isolated a lineage of Env apex-directed neutralizing antibodies, N90-VRC38.01-11, by using virus-like particles and conformationally stabilized Env trimers as B cell probes. A crystal structure of N90-VRC38.01 with a scaffolded V1V2 revealed a binding mode involving side-chain-to-side-chain interactions that reduced the distance the antibody loop must traverse the glycan shield, thereby facilitating V1V2 binding via a non-protruding loop. The N90-VRC38 lineage thus identifies a solution for V1V2-apex binding that provides a more conventional B cell pathway for vaccine design.

In situ turning defects of exfoliated Ti3C2 MXene into Fenton-like catalytic active sites.

Controllable in situ formation of nanoclusters with discrete active sites is highly desirable in heterogeneous catalysis. Herein, a titanium oxide-based Fenton-like catalyst is constructed using exfoliated Ti3C2 MXene as a template. Theoretical calculations reveal that a redox reaction between the surface Ti-deficit vacancies of the exfoliated Ti3C2 MXene and H2O2 molecules facilitates the in situ conversion of surface defects into titanium oxide nanoclusters anchoring on amorphous carbon (TiOx@C). The presence of mixed-valence Tiδ+ (δ = 0, 2, 3, and 4) within TiOx@C is confirmed by X-ray photoelectron spectroscopy (XPS) and X-ray absorption fine structure (XAFS) characterizations. The abundant surface defects within TiOx@C effectively promote the generation of reactive oxygen species (ROS) leading to superior and stable Fenton-like catalytic degradation of atrazine, a typical agricultural herbicide. Such an in situ construction of Fenton-like catalysts through defect engineering also applies to other MXene family materials, such as V2C and Nb2C.

Quantification of gallium cryo-FIB milling damage in biological lamellae.

Cryogenic electron microscopy (cryo-EM) can reveal the molecular details of biological processes in their native, cellular environment at atomic resolution. However, few cells are sufficiently thin to permit imaging with cryo-EM. Thinning of frozen cells to <500 nm lamellae by focused-ion-beam (FIB) milling has enabled visualization of cellular structures with cryo-EM. FIB milling represents a significant advance over prior approaches because of its ease of use, scalability, and lack of large-scale sample distortions. However, the amount of damage it causes to a thinned cell section has not yet been determined. We recently described an approach for detecting and identifying single molecules in cryo-EM images of cells using 2D template matching (2DTM). 2DTM is sensitive to small differences between a molecular model (template) and the detected structure (target). Here, we use 2DTM to demonstrate that under the standard conditions used for machining lamellae of biological samples, FIB milling introduces a layer of variable damage that extends to a depth of 60 nm from each lamella surface. This layer of damage limits the recovery of information for in situ structural biology. We find that the mechanism of FIB milling damage is distinct from radiation damage during cryo-EM imaging. By accounting for both electron scattering and FIB milling damage, we estimate that FIB milling damage with current protocols will negate the potential improvements from lamella thinning beyond 90 nm.

Generation of de novo miRNAs from template switching during DNA replication.

The mechanisms generating novel genes and genetic information are poorly known, even for microRNA (miRNA) genes with an extremely constrained design. All miRNA primary transcripts need to fold into a stem-loop structure to yield short gene products ([Formula: see text]22 nt) that bind and repress their mRNA targets. While a substantial number of miRNA genes are ancient and highly conserved, short secondary structures coding for entirely novel miRNA genes have been shown to emerge in a lineage-specific manner. Template switching is a DNA-replication-related mutation mechanism that can introduce complex changes and generate perfect base pairing for entire hairpin structures in a single event. Here, we show that the template-switching mutations (TSMs) have participated in the emergence of over 6,000 suitable hairpin structures in the primate lineage to yield at least 18 new human miRNA genes, that is 26% of the miRNAs inferred to have arisen since the origin of primates. While the mechanism appears random, the TSM-generated miRNAs are enriched in introns where they can be expressed with their host genes. The high frequency of TSM events provides raw material for evolution. Being orders of magnitude faster than other mechanisms proposed for de novo creation of genes, TSM-generated miRNAs enable near-instant rewiring of genetic information and rapid adaptation to changing environments.

Electrochemically active porous carbon nanospheres prepared by inhibition of pyrolytic condensation of polymers.

Porous carbon is a pivotal material for electrochemical applications. The manufacture of porous carbon has relied on chemical treatments (etching or template) that require processing in all areas of the carbon/carbon precursor. We present a unique approach to preparing porous carbon nanospheres by inhibiting the pyrolytic condensation of polymers. Specifically, the porous carbon nanospheres are obtained by coating a thin film of ZnO on polystyrene spheres. The porosity of the porous carbon nanospheres is controlled by the thickness of the ZnO shell, achieving a BET-specific area of 1,124 m2/g with a specific volume of 1.09 cm3/g. We confirm that under the support force by the ZnO shell, a hierarchical pore structure in which small mesopores are connected by large mesopores is formed and that the pore-associated sp3 defects are enriched. These features allow full utilization of the surface area of the carbon pores. The electrochemical capacitive performance of porous carbon nanospheres was evaluated, achieving a high capacitance of 389 F/g at 1 A/g, capacitance retention of 71% at a 20-fold increase in current density, and stability up to 30,000 cycles. In particular, we achieve a specific area-normalized capacitance of 34.6 µF/cm2, which overcomes the limitations of conventional carbon materials.

Structure-specific nucleases: role in Okazaki fragment maturation.

Proper function of structure-specific nucleases is key for faithful Okazaki fragment maturation (OFM) process completion. Deregulation of such nucleases leads to aberrant OFM and causes a spectrum of mutations, some of which may confer survival outcomes under specific stresses and serve as attractive targets for therapeutic intervention in human cancers.

Improved model quality assessment using sequence and structural information by enhanced deep neural networks.

Protein model quality assessment plays an important role in protein structure prediction, protein design and drug discovery. In this work, DeepUMQA2, a substantially improved version of DeepUMQA for protein model quality assessment, is proposed. First, sequence features containing protein co-evolution information and structural features reflecting family information are extracted to complement model-dependent features. Second, a novel backbone network based on triangular multiplication update and axial attention mechanism is designed to enhance information exchange between inter-residue pairs. On CASP13 and CASP14 datasets, the performance of DeepUMQA2 increases by 20.5 and 20.4% compared with DeepUMQA, respectively (measured by top 1 loss). Moreover, on the three-month CAMEO dataset (11 March to 04 June 2022), DeepUMQA2 outperforms DeepUMQA by 15.5% (measured by local AUC0,0.2) and ranks first among all competing server methods in CAMEO blind test. Experimental results show that DeepUMQA2 outperforms state-of-the-art model quality assessment methods, such as ProQ3D-LDDT, ModFOLD8, and DeepAccNet and DeepUMQA2 can select more suitable best models than state-of-the-art protein structure methods, such as AlphaFold2, RoseTTAFold and I-TASSER, provided themselves.

Genotype imputation accuracy and the quality metrics of the minor ancestry in multi-ancestry reference panels.

Large-scale imputation reference panels are currently available and have contributed to efficient genome-wide association studies through genotype imputation. However, whether large-size multi-ancestry or small-size population-specific reference panels are the optimal choices for under-represented populations continues to be debated. We imputed genotypes of East Asian (180k Japanese) subjects using the Trans-Omics for Precision Medicine reference panel and found that the standard imputation quality metric (Rsq) overestimated dosage r2 (squared correlation between imputed dosage and true genotype) particularly in marginal-quality bins. Variance component analysis of Rsq revealed that the increased imputed-genotype certainty (dosages closer to 0, 1 or 2) caused upward bias, indicating some systemic bias in the imputation. Through systematic simulations using different template switching rates (θ value) in the hidden Markov model, we revealed that the lower θ value increased the imputed-genotype certainty and Rsq; however, dosage r2 was insensitive to the θ value, thereby causing a deviation. In simulated reference panels with different sizes and ancestral diversities, the θ value estimates from Minimac decreased with the size of a single ancestry and increased with the ancestral diversity. Thus, Rsq could be deviated from dosage r2 for a subpopulation in the multi-ancestry panel, and the deviation represents different imputed-dosage distributions. Finally, despite the impact of the θ value, distant ancestries in the reference panel contributed only a few additional variants passing a predefined Rsq threshold. We conclude that the θ value substantially impacts the imputed dosage and the imputation quality metric value.

Flexibility of telomerase in binding the RNA template and DNA telomeric repeat.

Telomerase synthesizes telomeres at the ends of linear chromosomes by repeated reverse transcription from a short RNA template. Crystal structures of Tribolium castaneum telomerase reverse transcriptase (tcTERT) and cryoelectron microscopy (cryo-EM) structures of human and Tetrahymena telomerase have revealed conserved features in the reverse-transcriptase domain, including a cavity near the DNA 3' end and snug interactions with the RNA template. For the RNA template to translocate, it needs to be unpaired and separated from the DNA product. Here we investigate the potential of the structural cavity to accommodate a looped-out DNA bulge and enable the separation of the RNA/DNA hybrid. Using tcTERT as a model system, we show that a looped-out telomeric repeat in the DNA primer can be accommodated and extended by tcTERT but not by retroviral reverse transcriptase. Mutations that reduce the cavity size reduce the ability of tcTERT to extend the looped-out DNA substrate. In agreement with cryo-EM structures of telomerases, we find that tcTERT requires a minimum of 4 bp between the RNA template and DNA primer for efficient DNA synthesis. We also have determined the ternary-complex structure of tcTERT including a downstream RNA/DNA hybrid at 2.0-Å resolution and shown that a downstream RNA duplex, equivalent to the 5' template-boundary element in telomerase RNA, enhances the efficiency of telomere synthesis by tcTERT. Although TERT has a preformed active site without the open-and-closed conformational changes, it contains cavities to accommodate looped-out RNA and DNA. The flexible RNA-DNA binding likely underlies the processivity of telomeric repeat addition.

Template switching in DNA replication can create and maintain RNA hairpins.

The evolutionary origin of RNA stem structures and the preservation of their base pairing under a spontaneous and random mutation process have puzzled theoretical evolutionary biologists. DNA replication-related template switching is a mutation mechanism that creates reverse-complement copies of sequence regions within a genome by replicating briefly along either the complementary or nascent DNA strand. Depending on the relative positions and context of the four switch points, this process may produce a reverse-complement repeat capable of forming the stem of a perfect DNA hairpin or fix the base pairing of an existing stem. Template switching is typically thought to trigger large structural changes, and its possible role in the origin and evolution of RNA genes has not been studied. Here, we show that the reconstructed ancestral histories of RNA genes contain mutation patterns consistent with the DNA replication-related template switching. In addition to multibase compensatory mutations, the mechanism can explain complex sequence changes, although mutations breaking the structure rarely get fixed in evolution. Our results suggest a solution for the long-standing dilemma of RNA gene evolution and demonstrate how template switching can both create perfect stems with a single mutation event and help maintaining the stem structure over time. Interestingly, template switching also provides an elegant explanation for the asymmetric base pair frequencies within RNA stems.

A dynamic template complex mediates Munc18-chaperoned SNARE assembly.

Munc18 chaperones assembly of three membrane-anchored soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) into a four-helix bundle to mediate membrane fusion between vesicles and plasma membranes, leading to neurotransmitter or insulin release, glucose transporter (GLUT4) translocation, or other exocytotic processes. Yet, the molecular mechanism underlying chaperoned SNARE assembly is not well understood. Recent evidence suggests that Munc18-1 and Munc18-3 simultaneously bind their cognate SNAREs to form ternary template complexes - Munc18-1:Syntaxin-1:VAMP2 for synaptic vesicle fusion and Munc18-3:Syntaxin-4:VAMP2 for GLUT4 translocation and insulin release, which facilitate the binding of SNAP-25 or SNAP-23 to conclude SNARE assembly. Here, we further investigate the structure, dynamics, and function of the template complexes using optical tweezers. Our results suggest that the synaptic template complex transitions to an activated state with a rate of 0.054 s-1 for efficient SNAP-25 binding. The transition depends upon the linker region of syntaxin-1 upstream of its helical bundle-forming SNARE motif. In addition, the template complex is stabilized by a poorly characterized disordered loop region in Munc18-1. While the synaptic template complex efficiently binds both SNAP-25 and SNAP-23, the GLUT4 template complex strongly favors SNAP-23 over SNAP-25, despite the similar stabilities of their assembled SNARE bundles. Together, our data demonstrate that a highly dynamic template complex mediates efficient and specific SNARE assembly.