Biology subjects

Zhao, M.

Publications and source records attributed to Zhao, M..

At least 235 records · Page 13Linked to original sources

bioRxiv2020

Structural dynamics of the SARS-CoV-2 frameshift-stimulatory pseudoknot reveal topologically distinct conformers

The RNA pseudoknot that stimulates -1 programmed ribosomal frameshifting in SARS coronavirus-2 (SARS-CoV-2) is a possible drug target. To understand how this 3-stemmed pseudoknot responds to the mechanical tension applied by ribosomes during translation, which is thought to play a key role during frame-shifting, we probed its structural dynamics under tension using optical tweezers. Unfolding curves revealed that the frameshift signal formed multiple different structures: at least two distinct pseudoknotted conformers with different unfolding forces and energy barriers, as well as alternative stem-loop structures. Refolding curves showed that stem 1 formed first in the pseudoknotted conformers, followed by stem 3 and then stem 2. By extending the handle holding the RNA to occlude the 5' end of stem 1, the proportion of the different pseudoknot conformers could be altered systematically, consistent with structures observed in cryo-EM images and computational simulations that had distinct topologies: the 5' end of the RNA threaded through the 3-helix junction to form a ring-knot, or unthreaded as in more standard H-type pseudoknots. These results resolve the folding mechanism of the frameshift signal in SARS-CoV-2 and highlight the dynamic conformational heterogeneity of this RNA, with important implications for structure-based drug-discovery efforts.

biophysics↗

bioRxiv2020

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super-relaxed state: multiscale measurements and computational modeling

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, many in {beta}-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super-relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned hiPSC-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super-relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease. Significance StatementHeart disease is the leading cause of death worldwide, and hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, affecting over 1 in 200 people. Mutations in myosin, the motor protein responsible for contraction of the heart, are a common cause of HCM but have diverse effects on the biomechanics of the myosin protein. We demonstrate that complex biomechanical effects of mutations associated with heart disease can be effectively studied and understood using a multi-scale experimental and computational modeling approach. This work confirmed an important role for disruption of the super-relaxed state for one particular HCM mutation, and our approach can be extended to aid in the development of new targeted therapies for patients with different mutations.

biophysics↗

bioRxiv2020

Analysis of the Functional Differentiation Sites in Vertebrate Neuronal Nicotinic Acetylcholine Receptor Subunits

The nicotinic acetylcholine receptors (nAChR) belong to a large family of ligand-gated ion channels and are involved in the mediation of fast synaptic transmission. Each receptor is made up of five subunits that arrange symmetrically around a central pore. Despite the similarity in their sequences and structures, the properties of these subunits vary significantly. Thus, identifying the function-related sites specific to each subunit is essential for understanding the characteristics of the subunits and the receptors assembled by them. In this study, we examined the sequence features of the nine neuronal nAChRs subunits from twelve representative vertebrate species. Analysis revealed that all the subunits were subject to strong purifying selection in evolution, and each was under a unique pattern of selection pressures. At the same time, the functional constraints were not uniform within each subunit, with different domains in the molecule being subject to different selection pressures. Via evolutionary analyses, we also detected potential positive selection events in the subunits or subunit clusters, and identified the sites might be associated with the function specificity of each subunit. Furthermore, positive selection at some domains might contributed to the diversity of subunit function; for example, the {beta}9 strand might be related to the agonist specificity of subunit in heteromeric receptor and {beta}4-{beta}5 linker could be involved in Ca2+ permeability. Subunits 7, 4 and {beta}2 subunits possess a strong adaptability in vertebrates. Our results highlighted the importance of tracking functional differentiation in protein sequence underlying functional properties of nAChRs. In summary, our work may provide clues on understanding the diversity and the function specificity of the nAChR subunits, as well as the receptors co-assembled by them.

evolutionary biology↗

bioRxiv2020

Interaction Landscape of K29-Linked Ubiquitin Signaling Revealed by a Linkage-Specific Synthetic Antigen-Binding Fragment

Protein ubiquitination shows remarkable topological and functional diversity through the polymerization of ubiquitin via different linkages. Deciphering the cellular ubiquitin code is of central importance to understand the physiology of the cell. Among the eight possible linkages, K29-linked polyubiquitin is a relatively abundant type of polyubiquitin in both human and yeast cells. However, our understanding of its function is rather limited due to the lack of specific binders as tools to detect K29-linked polyubiquitin. In this study, we screened and characterized a synthetic antigen-binding fragment, termed sAB-K29, that can specifically recognize K29-linked polyubiquitin using chemically synthesized K29-linked diubiquitin. We further determined the crystal structure of this fragment bound to the K29-linked diubiquitin, which revealed the molecular basis of specificity. Using sAB-K29 as a tool, we uncovered that K29-linked ubiquitination is involved in different kinds of cellular proteotoxic stress response as well as cell cycle regulation. In particular, we showed that K29-linked ubiquitination is enriched in the midbody and downregulation of the K29-linked ubiquitination signal arrests cells in G1/S phase.

cell biology↗

bioRxiv2020

Antimicrobial emulsifier - glycerol monolaurate impacts gut micobiome inducing distinct effects on metabolic syndrome in low-fat diet fed mice

Previous study demonstrated 150 mg{middle dot}kg-1 glycerol monolaurate (GML) consumption induced gut microbiota dysbiosis and metabolic syndrome (MetS) in low-fat diet fed mice. However, little is known about the dose-effect of dietary GML modulating the gut microbiome alterations and its impacts on the induction of MetS in low-fat diet. Dietary GML-induced effects on MetS and gut microbiota alterations were investigated, combined with antibiotics-treated germ-free experiment and specific gut microbiota transplantation. Our results demonstrated that high-dose (500 mg{middle dot}kg-1) GML alleviated MetS by significantly decreasing the body weight, weight gain, food intake, fat droplet size and percentage of abdominal fat, serum triglycerides (TG), LDL, LPS, TNF- and atherogenic index, compared to the medium-dose (150 mg{middle dot}kg-1) GML. Importantly, high-dose GML significantly increased Lactobacillus reuteri compared to the medium-dose GML. Co-occurrence network analysis revealed Lactobacillus reuteri was positively associated with the metabolic improvement of high-dose GML. Notably, antibiotics-treated germ-free experiment and Lactobacillus reuteri transplantation demonstrated that altered gut microbiota was necessary and sufficient for GML-induced distinct effects on metabolic syndrome. Our results indicate that GML impacts gut microbiome inducing distinct effects on metabolic syndrome, thereby calling for reassessing the safe dosage of GML and other non-specific antibacterial food additives. IMPORTANCEGrowing evidence indicate that the broad use of food emulsifying agents may lead to increase the societal incidence of obesity/ MetS and other chronic inflammatory diseases. GML is widely and regularly consumed as a generally safe food emulsifier and as a potent antimicrobial agent in commonly foods such as meat products, cereals and soft beverage by the general public. Our results indicate that GML impacts gut microbiome inducing distinct effects on metabolic syndrome. Our study provides important and timely evidence supporting the emerging concept that non-specific antibacterial food additives have two-sided effect on gut microbiota contributing to the uncertainties for the incidence of obesity/metabolic syndrome and other chronic inflammatory diseases.

microbiology↗

bioRxiv2020

Cevipabulin-tubulin complex reveals a novel agent binding site on α-tubulin and provides insights into microtubule dynamic instability

Microtubule, composed of {beta}-tubulin heterodimers, remains as one of the most popular anticancer targets for decades. To date, anti-microtubule drugs are mainly functionally divided into microtubule-destabilizing and microtubule-stabilizing agents while microtubule- or tubulin-degradation agents are rarely reported. Six known binding sites on tubulin dimer are identified with five sites on {beta}-tubulin and only one site on -tubulin (pironetin site), hinting compounds binding to -tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our X-ray crystallography study reveals that, in addition binding to the vinblastine site, cevipabulin also binds to a novel site on -tubulin (named the seventh site) which located near the nonexchangeable GTP. Interestingly, we find the binding of cevipabulin to the seventh site induces tubulin degradation. As the non-exchangeable GTP has structural role and is important for the stability of tubulin dimers, we propose and confirm the tubulin degradation mechanism as: Cevipabulin at the seventh site puts the T5 loop outward to make the non-exchangeable GTP exchangeable, which reduces the stability of tubulin and results in its destabilization and degradation. Our results confirm a novel agent binding site on -tubulin and shed light on the development of tubulin degraders as a new generation of anti-microtubule drugs targeting this novel site.

cell biology↗

bioRxiv2020

Chromosome-level Genome Assembly of a Regenerable Maize Inbred Line A188

The highly embryogenic and transformable maize inbred line A188 is an attractive model for analyzing maize gene function. Here we constructed a chromosome-level genome assembly of A188 using long reads and optical maps. Genome comparison of A188 with the reference line B73 identified pervasive structural variation, including a 1.8 Mb duplication on the Gametophyte factor1 locus for unilateral cross-incompatibility and six inversions of 0.7 Mb or greater. Increased copy number of the gene, carotenoid cleavage dioxygenase 1 (ccd1) in A188 is associated with elevated expression during seed development. High ccd1 expression together with low expression of yellow endosperm 1 (y1) condition reduced carotenoid accumulation, which accounts for the white seed phenotype of A188 that contrasts with the yellow seed of B73 that has high expression of y1 and low expression of the single-copy ccd1. Further, transcriptome and epigenome analyses with the A188 reference genome revealed enhanced expression of defense pathways and altered DNA methylation patterns of embryonic callus.

genomics↗

bioRxiv2020

Human genetic variants disrupt RGS14 nuclear shuttling and regulation of LTP in hippocampal neurons

The human genome contains vast genetic diversity in the form of naturally occurring coding variants, yet the impact of these variants on protein function and physiology is poorly understood. RGS14 is a multifunctional signaling protein that suppresses synaptic plasticity in dendritic spines of hippocampal neurons. RGS14 also is a nucleocytoplasmic shuttling protein, suggesting that balanced nuclear import/export and dendritic spine localization are essential for RGS14 functions. We identified genetic variants L505R (LR) and R507Q (RQ) located within the nuclear export sequence (NES) of human RGS14. Here we report that RGS14 carrying LR or RQ profoundly impacts protein functions in hippocampal neurons and brain. Following nuclear import, RGS14 nuclear export is regulated by Exportin 1 (XPO1/CRM1). Remarkably, LR and RQ variants disrupt RGS14 binding to Gi1-GDP and XPO1, nucleocytoplasmic equilibrium, and capacity to inhibit LTP. Variant LR accumulates irreversibly in the nucleus, preventing RGS14 binding to G proteins, localization to dendritic spines, and inhibitory actions on LTP induction, while variant RQ exhibits a mixed phenotype. When introduced into mice by CRISPR/Cas9, RGS14-LR protein expression was detected predominantly in the nuclei of neurons within hippocampus, central amygdala, piriform cortex, and striatum, brain regions associated with learning and synaptic plasticity. Whereas mice completely lacking RGS14 exhibit enhanced spatial learning, mice carrying variant LR exhibit normal spatial learning, suggesting that RGS14 may have distinct functions in the nucleus independent from those in dendrites and spines. These findings show that naturally occurring genetic variants can profoundly alter normal protein function, impacting physiology in unexpected ways.

molecular biology↗

bioRxiv2020

Multi-modal pooled Perturb-CITE-Seq screens in patient models define novel mechanisms of cancer immune evasion

Resistance to immune checkpoint inhibitors (ICI) that activate T cell mediated anti-tumor immunity is a key challenge in cancer therapy, yet the underlying mechanisms remain poorly understood. To further elucidate those, we developed a new approach, Perturb-CITE-seq, for pooled CRISPR perturbation screens with multi-modal RNA and protein single-cell profiling readout and applied it to screen patient-derived autologous melanoma and tumor infiltrating lymphocyte (TIL) co-cultures. We profiled RNA and 20 surface proteins in over 218,000 cells under ~750 perturbations, chosen by their membership in an immune evasion program that is associated with immunotherapy resistance in patients. Our screen recovered clinically-relevant resistance mechanisms concordantly reflected in RNA, protein and perturbation effects on susceptibility to T cell mediated killing. These were organized in eight co-functional modules whose perturbation distinctly affect four co-regulated programs associated with immune evasion. Among these were defects in the IFN{gamma}-JAK/STAT pathway and in antigen presentation, and several novel mechanisms, including loss or downregulation of CD58, a surface protein without known mouse homolog. Leveraging the rich profiles in our screen, we found that loss of CD58 did not compromise MHC protein expression and that CD58 was not transcriptionally induced by the IFN{gamma} pathway, allowing us to distinguish it as a novel mechanism of immune resistance. We further show that loss of CD58 on cancer cells conferred immune evasion across multiple T cell and Natural Killer cell patient co-culture models. Notably, CD58 is downregulated in tumors with resistance to immunotherapy in melanoma patients. Our work identifies novel mechanisms at the nexus of immune evasion and drug resistance and provides a general framework for deciphering complex mechanisms by large-scale perturbation screens with multi-modal singlecell profiles, including in systems consisting of multiple cell types.

genomics↗

bioRxiv2020

BNIP3 phosphorylation by JNK1/2 promotes mitophagy via enhancing its stability under hypoxia

Mitophagy is an important metabolic mechanism that modulates mitochondrial quality and quantity by selectively removing damaged or unwanted mitochondria. BNIP3, a mitochondrial outer membrane protein, is a mitophagy receptor that mediates mitophagy under various stresses, particularly hypoxia, since BNIP3 is a hypoxia-responsive protein. However, the underlying mechanisms that regulate BNIP3 and thus mediate mitophagy under hypoxic conditions remain elusive. Here, we demonstrate that in hypoxia JNK1/2 phosphorylates BNIP3 at Ser 60/Thr 66, which hampers proteasomal degradation of BNIP3 and drives mitophagy by facilitating the direct binding of BNIP3 to LC3, while PP1/2A represses mitophagy by dephosphorylating BNIP3 and triggering its proteasomal degradation. These findings reveal the intrinsic mechanisms cells use to regulate mitophagy via the JNK1/2-BNIP3 pathway in response to hypoxia. Thus, the JNK1/2-BNIP3 signaling pathway strongly links mitophagy to hypoxia and may be a promising therapeutic target for hypoxia-related diseases.

cell biology↗

bioRxiv2020

Dach1 extends artery networks and protects against cardiac injury

Coronary artery disease (CAD) is the leading cause of death worldwide, but there are currently no available methods to stimulate growth or regeneration of artery networks in diseased hearts. Studying how arteries are built during embryonic development could illuminate strategies for re-building these vessels in the setting of ischemic heart disease. We previously found, using loss-of-function experiments, that the transcription factor Dach1 is required for coronary artery development in mouse embryos. Here, we report that Dach1 overexpression in endothelial cells (ECs) extended coronary arteries and improved survival and heart function in adult mice following myocardial infarction (MI). Dach1 overexpression increased the length and number of arterial end branches, in both heart and retinal vasculature, by causing additional capillary ECs to differentiate into arterial ECs and contribute to growing arteries. Single-cell RNA sequencing (scRNAseq) of ECs undergoing Dach1-induced arterial specification indicated that it potentiated normal artery differentiation, rather than functioning as a master regulator of artery cell fate. ScRNAseq also showed that normal arterial differentiation is accompanied by repression of lipid metabolism genes, which were also repressed by Dach1 prior to arterialization. Together, these results demonstrate that increasing the expression level of Dach1 is a novel pathway for driving specification of artery ECs and extending arterial vessels, which could be explored as a means of increasing artery coverage to mitigate the effects of CAD.

developmental biology↗

bioRxiv2020

PI3K inhibition reverses migratory direction of single cells but not cell groups in electric field

Motile cells migrate directionally in the electric field in a process known as galvanotaxis. Galvanotaxis is important in wound healing, development, cell division, and nerve growth. Different cell types migrate in opposite directions in electric fields, to either cathode, or anode, and the same cell can switch the directionality depending on chemical conditions. We previously reported that individual fish keratocyte cells sense electric fields and migrate to the cathode, while inhibition of PI3K reverses single cells to the anode. Many physiological processes rely on collective, not individual, cell migration, so here we report on directional migration of cohesive cell groups in electric fields. Uninhibited cell groups of any size move to the cathode, with speed decreasing and directionality increasing with the group size. Surprisingly, large groups of PI3K-inhibited cells move to the cathode, in the direction opposite to that of individual cells, which move to the anode, while such small groups are not persistently directional. In the large groups, cells velocities are distributed unevenly: the fastest cells are at the front of the uninhibited groups, but at the middle and rear of the PI3K-inhibited groups. Our results are most consistent with the hypothesis, supported by the computational model, that cells inside and at the edge of the groups interpret directional signals differently. Namely, cells in the group interior are directed to the cathode independently of their chemical state. Meanwhile, edge cells behave like the individual cells: they are directed to the cathode/anode in uninhibited/PI3K-inhibited groups, respectively. As a result, all cells drive uninhibited groups to the cathode, but a mechanical tug-of-war between the inner and edge cells directs large PI3K-inhibited groups with cell majority in the interior to the cathode, while rendering small groups non-directional. Significance statementMotile cells migrate directionally in electric fields. This behavior - galvanotaxis - is important in many physiological phenomena. Individual fish keratocytes migrate to the cathode, while inhibition of PI3K reverses single cells to the anode. Uninhibited cell groups move to the cathode. Surprisingly, large groups of PI3K-inhibited cells also move to the cathode, in the direction opposite to that of individual cells. The fastest cells are at the front of the uninhibited groups, but at the middle and rear of the PI3K-inhibited groups. We posit that inner and edge cells interpret directional signals differently, and that a tug-of-war between the edge and inner cells directs the cell groups. These results shed light on general principles of collective cell migration.

biophysics↗

bioRxiv2020

Resistance to tembotrione, a 4-hydroxyphenylpyruvate dioxygenase-inhibitor, in Sorghum bicolor

Postemergence grass weed control continues to be a big challenge in grain sorghum (Sorghum bicolor L. Moench), primarily due to a lack of herbicide options registered for use in this crop. The development of herbicide-resistant sorghum technology to facilitate broad-spectrum postemergence weed control is an economical and viable solution. The 4-hydroxyphenylpyruvate dioxygenase-inhibitor herbicides (e.g. mesotrione or tembotrione) can control broad-spectrum of weeds including grasses, which, however, is not registered for postemergence application in sorghum due to crop injury. In this study we identified two tembotrione-resistant sorghum genotypes (G-200, G-350) and one highly susceptible genotype (S-1) through screening 317 sorghum lines from the sorghum association panel (SAP). Compared to S-1, G-200 and G-350 exhibited 10- and 7-fold more resistance to tembotrione, respectively. Genetic analyses of the F1 and F2 progeny generated from a cross between tembotrione-resistant and -susceptible genotypes demonstrated that the resistance is a semi-dominant polygenic trait. Furthermore, cytochrome P450 (CYP)-inhibitor assay using malathion and piperonyl butoxide suggested possible CYP-mediated metabolism of tembotrione in G-200 and G-350. Genotype-by-sequencing based quantitative trait loci (QTL) mapping revealed eight QTLs associated with tembotrione resistance in grain sorghum. Sorghum genotypes G-200 and G-350 confer a high level of metabolic resistance to tembotrione and controlled by a polygenic trait. There is an enormous potential to introgress the tembotrione resistance into breeding lines to develop agronomically desirable sorghum hybrids. One-sentence summaryThis research focuses on characterization, genetic analyses, identification of QTLs-linked to metabolic resistance to tembotrione in Sorghum bicolor, for improved weed control and increased yield

plant biology↗

bioRxiv2020

Mechanically stable knot formed by strand threading in Zika virus RNA confers RNase resistance

Exoribonuclease-resistant RNAs (xrRNAs) from viruses prevent digestion by host exoribonucleases, creating sub-genomic viral RNAs that can enhance infection and pathogenicity. Novel knotted structures in xrRNAs are proposed to act as mechanical road-blocks to RNases. Studying an xrRNA from Zika virus with optical tweezers, we found that it was the most mechanically stable RNA structure yet observed. The knot folded by threading the 5′ end into the cleft of a Mg2+-coordinated three-helix junction before pseudoknot interactions closed a ring around it. Both the threading and pseudoknot were required to generate the extremely force-resistant knot, whose formation correlated directly with RNase resistance both in the wild-type xrRNA and a low-resistance mutant. This work clarifies the folding and mechanism of action of an important new class of RNA.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biophysics↗

bioRxiv2020

Anti-frameshifting ligand active against SARS coronavirus-2 is resistant to natural mutations of the frameshift-stimulatory pseudoknot

The coronavirus SARS-CoV-2 causing the COVID-19 pandemic uses −1 programmed ribosomal frameshifting (−1 PRF) to control the expression levels of key viral proteins. Because modulating −1 PRF can attenuate viral propagation, ligands binding to the viral RNA pseudoknot that stimulates −1 PRF may prove useful as therapeutics. Mutations in the pseudoknot have been observed over the course of the pandemic, but how they affect −1 PRF and the activity of inhibitors is unknown. Cataloguing natural mutations in all parts of the SARS-CoV-2 pseudoknot, we studied a panel of 6 mutations in key structural regions. Most mutations left the −1 PRF efficiency unchanged, even when base-pairing was disrupted, but one led to a remarkable three-fold decrease, suggesting that SARS-CoV-2 propagation may be less sensitive to modulation of −1 PRF efficiency than some other viruses. Examining the effects of one of the few small-molecule ligands known to suppress −1 PRF significantly in SARS-CoV, we found that it did so by similar amounts in all SARS-CoV-2 mutants tested, regardless of the basal −1 PRF efficiency, indicating that the activity of anti-frameshifting ligands can be resistant to natural pseudoknot mutations. These results have important implications for therapeutic strategies targeting SARS-CoV-2 through modulation of −1 PRF.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Machine learning-based promoter strength prediction derived from a fine-tuned synthetic promoter library in Escherichia coli

Promoters are one of the most critical regulatory elements controlling metabolic pathways. However, in recent years, researchers have simply perfected promoter strength, but ignored the relationship between the internal sequences and promoter strength. In this context, we constructed and characterized a mutant promoter library of Ptrc through dozens of mutation-construction-screening-characterization engineering cycles. After excluding invalid mutation sites, we established a synthetic promoter library, which consisted of 3665 different variants, displaying an intensity range of more than two orders of magnitude. The strongest variant was 1.52-fold stronger than a 1 mM isopropyl-{beta}-D-thiogalactoside driven PT7 promoter. Our synthetic promoter library exhibited superior applicability when expressing different reporters, in both plasmids and the genome. Different machine learning models were built and optimized to explore relationships between the promoter sequences and transcriptional strength. Finally, our XgBoost model exhibited optimal performance, and we utilized this approach to precisely predict the strength of artificially designed promoter sequences. Our work provides a powerful platform that enables the predictable tuning of promoters to achieve the optimal transcriptional strength.

synthetic biology↗

bioRxiv2020

Modeling the structure of the frameshift stimulatory pseudoknot in SARS-CoV-2 reveals multiple possible conformers

The coronavirus causing the COVID-19 pandemic, SARS-CoV-2, uses -1 programmed ribosomal frameshifting (-1 PRF) to control the relative expression of viral proteins. As modulating -1 PRF can inhibit viral replication, the RNA pseudoknot stimulating -1 PRF may be a fruitful target for therapeutics treating COVID-19. We modeled the unusual 3-stem structure of the stimulatory pseudoknot of SARS-CoV-2 computationally, using multiple blind structural prediction tools followed by s-long molecular dynamics simulations. The results were compared for consistency with nuclease-protection assays and single-molecule force spectroscopy measurements of the SARS-CoV-1 pseudoknot, to determine the most likely conformations. We found several possible conformations for the SARS-CoV-2 pseudoknot, all having an extended stem 3 but with different packing of stems 1 and 2. Several conformations featured rarely-seen threading of a single strand through the junction formed between two helices. These structural models may help interpret future experiments and support efforts to discover ligands inhibiting -1 PRF in SARS-CoV-2.

biophysics↗

bioRxiv2020

Genus-wide characterization of bumblebee genomes reveals variation associated with key ecological and behavioral traits of pollinators

Bumblebees are a diverse group of globally important pollinators in natural ecosystems and for agricultural food production. With both eusocial and solitary lifecycle phases, and some social parasite species, they are especially interesting models to understand social evolution, behavior, and ecology. Reports of many species in decline point to pathogen transmission, habitat loss, pesticide usage, and global climate change, as interconnected causes. These threats to bumblebee diversity make our reliance on a handful of well-studied species for agricultural pollination particularly precarious. To broadly sample bumblebee genomic and phenotypic diversity, we de novo sequenced and assembled the genomes of 17 species, representing all 15 subgenera, producing the first genus-wide quantification of genetic and genomic variation potentially underlying key ecological and behavioral traits. The species phylogeny resolves subgenera relationships while incomplete lineage sorting likely drives high levels of gene tree discordance. Five chromosome-level assemblies show a stable 18-chromosome karyotype, with major rearrangements creating 25 chromosomes in social parasites. Differential transposable element activity drives changes in genome sizes, with putative domestications of repetitive sequences influencing gene coding and regulatory potential. Dynamically evolving gene families and signatures of positive selection point to genus-wide variation in processes linked to foraging, diet and metabolism, immunity and detoxification, as well as adaptations for life at high altitudes. These high-quality genomic resources capture natural genetic and phenotypic variation across bumblebees, offering new opportunities to advance our understanding of their remarkable ecological success and to identify and manage current and future threats.

genomics↗