ABSTRACT
Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in lipid droplet (LD) biogenesis and in regulating LD morphology, pathogenic variants of which are associated with Berardinelli-Seip congenital generalized lipodystrophy type 2 (BSCL2). To model BSCL2 disease, we generated an orthologous BSCL2 variant, seip-1(A185P), in Caenorhabditis elegans. In this study, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P), including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, lmbr-1, which is an ortholog of human limb development membrane protein 1 (LMBR1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(S647F) and lmbr-1(P314L), both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.
Subject(s)
GTP-Binding Protein gamma Subunits , Heterotrimeric GTP-Binding Proteins , Lipodystrophy, Congenital Generalized , Lipodystrophy , Animals , Humans , Lipodystrophy, Congenital Generalized/genetics , Lipodystrophy, Congenital Generalized/metabolism , Heterotrimeric GTP-Binding Proteins/genetics , Heterotrimeric GTP-Binding Proteins/metabolism , GTP-Binding Protein gamma Subunits/genetics , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Lipodystrophy/geneticsABSTRACT
The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole-genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]), significantly increased brood size and ovulation rate, as well as alleviating the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Expression of GEX-3 in the soma is required to rescue the brood size defects in pezo-1(R2405P) animals. Actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the PIEZO coordinates with the cytoskeleton regulator to maintain the F-actin network and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.
Subject(s)
Actins , Arthrogryposis , Ophthalmoplegia , Retinal Diseases , Animals , Female , Humans , Actins/genetics , Arthrogryposis/genetics , Caenorhabditis elegans/genetics , Ion Channels , Mutation/genetics , PolymerizationABSTRACT
Maintaining the metabolic homeostasis of fatty acids is crucial for human health. Excess fatty acids are stored in lipid droplets (LDs), the primary energy reservoir that helps regulate fat and lipid homeostasis in nearly all cell types. Seipin (BSCL2), a conserved endoplasmic reticulum protein, plays a critical role in LD biogenesis and regulating LD morphology. Pathogenic variants of seipin are associated with multiple human genetic diseases, including Berardinelli-Seip Congenital Generalized Lipodystrophy Type 2 (BSCL2). However, the cellular and molecular mechanisms by which dysfunctional seipin leads to these diseases remain unclear. To model BSCL2 disease, we generated an orthologous BSCL2 pathogenic variant seip-1(A185P) using CRISPR/Cas9 genome editing in Caenorhabditis elegans . This variant led to severe developmental and cellular defects, including embryonic lethality, impaired eggshell formation, and abnormally enlarged LDs. We set out to identify genetic determinants that could suppress these defective phenotypes in the seip-1(A185P) mutant background. To this end, we conducted an unbiased chemical mutagenesis screen to identify genetic suppressors that restore embryonic viability in the seip-1(A185P) mutant background. A total of five suppressor lines were isolated and recovered from the screen. The defective phenotypes of seip-1(A185P) , including embryonic lethality and impaired eggshell formation, were significantly suppressed in each suppressor line. Two of the five suppressor lines also alleviated the enlarged LDs in the oocytes. We then mapped a suppressor candidate gene, R05D3.2 (renamed as lmbr-1 ), which is an ortholog of human LMBR1 (limb development membrane protein 1). The CRISPR/Cas9 edited lmbr-1 suppressor alleles, lmbr-1(Ser647Phe) and lmbr-1(Pro314Leu) , both significantly suppressed embryonic lethality and defective eggshell formation in the seip-1(A185P) background. The newly identified suppressor lines offer valuable insights into potential genetic interactors and pathways that may regulate seipin in the lipodystrophy model.
ABSTRACT
Sangivamycin (S) is an adenosine (A) nucleoside analog with low nanomolar antiviral activity against SARS-CoV-2 in vitro. Previously, low nanomolar antiviral efficacy was revealed when tested against multiple viral variants in several cell types. SARS-CoV-2 RNA isolated from live virus infected cells and the virions released from these cells was analyzed by mass spectrometry (MS) for S incorporation. Dose-dependent incorporation occurred up to 1.8 S per 1,000 nucleotides (49 S per genome) throughout the viral genomes isolated from both infected cells and viral particles, but this incorporation did not change the viral mutation rate. In contrast, host mRNA, affinity purified from the same infected and treated cells, contained little or no S. Sangivamycin triphosphate (STP) was synthesized to evaluate its incorporation into RNA by recombinant SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) under defined in vitro conditions. SARS-CoV-2 RdRp showed that S was not a chain terminator and S containing oligonucleotides templated as A. Though the antiviral mechanism remains to be determined, the data suggests that SARS-CoV-2 RdRp incorporates STP into SARS-CoV-2 RNA, which does not significantly impair viral RNA synthesis or the mutation rate.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , RNA, Viral/genetics , RNA-Dependent RNA Polymerase/metabolism , Antiviral Agents/chemistryABSTRACT
The mechanosensitive PIEZO channel family has been linked to over 26 disorders and diseases. Although progress has been made in understanding these channels at the structural and functional levels, the underlying mechanisms of PIEZO-associated diseases remain elusive. In this study, we engineered four PIEZO-based disease models using CRISPR/Cas9 gene editing. We performed an unbiased chemical mutagen-based genetic suppressor screen to identify putative suppressors of a conserved gain-of-function variant pezo-1[R2405P] that in human PIEZO2 causes distal arthrogryposis type 5 (DA5; p. R2718P). Electrophysiological analyses indicate that pezo-1(R2405P) is a gain-of-function allele. Using genomic mapping and whole genome sequencing approaches, we identified a candidate suppressor allele in the C. elegans gene gex-3. This gene is an ortholog of human NCKAP1 (NCK-associated protein 1), a subunit of the Wiskott-Aldrich syndrome protein (WASP)-verprolin homologous protein (WAVE/SCAR) complex, which regulates F-actin polymerization. Depletion of gex-3 by RNAi, or with the suppressor allele gex-3(av259[L353F]) , significantly restored the small brood size and low ovulation rate, as well as alleviated the crushed oocyte phenotype of the pezo-1(R2405P) mutant. Auxin-inducible degradation of GEX-3 revealed that only somatic-specific degradation of GEX-3 restored the reduced brood size in the pezo-1(R2405P) mutants. Additionally, actin organization and orientation were disrupted and distorted in the pezo-1 mutants. Mutation of gex-3(L353F) partially alleviated these defects. The identification of gex-3 as a suppressor of the pathogenic variant pezo-1(R2405P) suggests that the cytoskeleton plays an important role in regulating PIEZO channel activity and provides insight into the molecular mechanisms of DA5 and other PIEZO-associated diseases.
ABSTRACT
Germline stem cell proliferation in C. elegans requires activation of the GLP-1/Notch receptor, which is located on the germline plasma membrane and encoded by the glp-1 gene. We previously identified several genes whose products directly or indirectly promote activity of the GLP-1 signaling pathway by finding mutations that enhance the germline phenotype of a glp-1(ts) allele, glp-1(bn18) . Here, we report phenotypic and molecular analysis of a new ekl-1 allele, ekl-1(om92) , that enhances the glp-1(bn18) phenotype. ekl-1(om92) is a 244 bp deletion predicted to generate a frameshift and premature termination codon, yielding a severely truncated protein, suggesting it is a null allele.
ABSTRACT
Geneticists approach biology with a simple question: which genes are required for the pathway or process of interest? Classical genetic screens (aka forward genetics) in model organisms such as Caenorhabditis elegans have been the method of choice for answering that question. Next-generation sequencing provides the means to generate a comprehensive list of sequence variants, including the mutation of interest. Herein is described a workflow for sample preparation and data analysis to allow the simultaneous mapping and identification of candidate mutations by whole-genome sequencing in Caenorhabditis elegans.
Subject(s)
Caenorhabditis elegans , DNA Mutational Analysis , High-Throughput Nucleotide Sequencing , Whole Genome Sequencing , Animals , Caenorhabditis elegans/genetics , DNA Mutational Analysis/methods , High-Throughput Nucleotide Sequencing/methods , MutationABSTRACT
BACKGROUND: The objective of the authors was to assess the relationships between tobacco smoke exposure (TSE) and dental health and dental care visits among US children. METHODS: The authors examined 2018-2019 National Survey of Children's Health data on TSE, dental health, and oral health care visits. Children aged 1 through 11 years (N = 32,214) were categorized into TSE groups: no home TSE (did not live with a smoker), thirdhand smoke (THS) exposure (lived with a smoker who did not smoke inside the home), or secondhand smoke (SHS) and THS exposure (lived with a smoker who smoked inside the home). The authors conducted multivariable logistic regression analyses, adjusting for child age, sex, race or ethnicity, prematurity, caregiver education level, family structure, and federal poverty threshold. RESULTS: Children with home SHS and THS exposure were at increased odds of having frequent or chronic difficulty with 1 or more oral health problem (adjusted odds ratio [AOR], 1.59; 95% CI, 1.07 to 2.35; P = .022) and carious teeth or caries (AOR, 1.74; 95% CI 1.14 to 2.65; P = .010) than those with no TSE. Compared with children aged 1 through 11 years with no TSE, children with SHS and THS exposure were 2.22 times (95% CI, 1.01 to 4.87; P = .048) more likely to have not received needed oral health care but at decreased odds of having had any kind of oral health care visit (AOR, 0.55; 95% CI, 0.32 to 0.95; P = .032), including a preventive oral health care visit (AOR, 0.60; 95% CI, 0.36 to 0.99; P = .047). CONCLUSIONS: TSE in children is associated with caries and inadequate oral health care visits. PRACTICAL IMPLICATIONS: The pediatric dental visit is an opportune time to educate caregivers who smoke about dental health to improve their children's teeth condition and increase oral health care visits.
Subject(s)
Dental Caries , Tobacco Smoke Pollution , Child , Child, Preschool , Delivery of Health Care , Dental Caries/epidemiology , Dental Caries/etiology , Ethnicity , Family , Humans , Infant , Tobacco Smoke Pollution/adverse effects , Tobacco Smoke Pollution/analysisABSTRACT
Sangivamycin is a nucleoside analog that is well tolerated by humans and broadly active against phylogenetically distinct viruses, including arenaviruses, filoviruses, and orthopoxviruses. Here, we show that sangivamycin is a potent antiviral against multiple variants of replicative severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with half-maximal inhibitory concentration in the nanomolar range in several cell types. Sangivamycin suppressed SARS-CoV-2 replication with greater efficacy than remdesivir (another broad-spectrum nucleoside analog). When we investigated sangivamycin's potential for clinical administration, pharmacokinetic; absorption, distribution, metabolism, and excretion (ADME); and toxicity properties were found to be favorable. When tested in combination with remdesivir, efficacy was additive rather than competitive against SARS-CoV-2. The proven safety in humans, long half-life, potent antiviral activity (compared to remdesivir), and combinatorial potential suggest that sangivamycin is likely to be efficacious alone or in combination therapy to suppress viremia in patients. Sangivamycin may also have the ability to help combat drug-resistant or vaccine-escaping SARS-CoV-2 variants since it is antivirally active against several tested variants. Our results support the pursuit of sangivamycin for further preclinical and clinical development as a potential coronavirus disease 2019 therapeutic.
Subject(s)
Antiviral Agents , Pyrimidine Nucleosides , SARS-CoV-2/drug effects , Animals , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Antiviral Agents/toxicity , COVID-19/virology , Cell Line, Tumor , Cell Survival/drug effects , Chlorocebus aethiops , Female , Humans , Male , Mice , Pyrimidine Nucleosides/pharmacokinetics , Pyrimidine Nucleosides/pharmacology , Pyrimidine Nucleosides/toxicity , Vero CellsABSTRACT
RNA represents a potential target for new antiviral therapies, which are urgently needed to address public health threats such as the human immunodeficiency virus (HIV). We showed previously that the interaction between the viral Tat protein and the HIV-1 trans-activation response (TAR) RNA was blocked by TB-CP-6.9a. This cyclic peptide was derived from a TAR-binding loop that emerged during lab evolution of a TAR-binding protein (TBP) family. Here we synthesized and characterized a next-generation, cyclic-peptide library based on the TBP scaffold. We sought to identify conserved RNA-binding interactions and the influence of cyclization linkers on RNA binding and antiviral activity. A diverse group of cyclization linkers, encompassing disulfide bonds to bicyclic aromatic staples, was used to restrain the cyclic peptide geometry. Thermodynamic profiling revealed specific arginine-rich sequences with low to submicromolar affinity driven by enthalpic and entropic contributions. The best compounds exhibited no appreciable off-target binding to related molecules, such as BIV TAR and human 7SK RNAs. A specific arginine-to-lysine change in the highest affinity cyclic peptide reduced TAR binding by tenfold, suggesting that TBP-derived cyclic peptides use an arginine-fork motif to recognize the TAR major groove while differentiating the mode of binding from other TAR-targeting molecules. Finally, we showed that HIV infectivity in cell culture was reduced in the presence of cyclic peptides constrained by methylene or naphthalene-based linkers. Our findings provide insight into the molecular determinants required for HIV-1 TAR recognition and antiviral activity. These findings are broadly relevant to the development of antivirals that target RNA molecules.
Subject(s)
Antiviral Agents/chemistry , HIV-1/chemistry , Peptides, Cyclic/chemistry , RNA, Viral/chemistry , HEK293 Cells , HIV Infections/drug therapy , HIV Infections/genetics , HIV Infections/metabolism , HIV-1/genetics , HIV-1/metabolism , Humans , Protein Binding , RNA, Viral/genetics , RNA, Viral/metabolismABSTRACT
The C. elegans dauer is an alternative third stage larva induced by dense population and adverse environmental conditions. Genes whose mutants caused dauer formation constitutive (Daf-c) and dauer formation defective (Daf-d) phenotypes were ordered via epistasis into a signaling network, with upstream DAF-7/TGF-beta and DAF-11/receptor guanylyl cyclase defining sensory branches and downstream DAF-2/Insulin receptor and DAF-12/nuclear hormone receptor executing the dauer decision. Mutations in the Scd genes were defined as incompletely penetrant suppressors of the constitutive dauer phenotype conferred by mutation of the DAF-7/TGF-beta signaling axis. SCD-2 was previously shown to be an ortholog of mammalian ALK (Anaplastic Lymphoma Kinase), a receptor tyrosine kinase. Mutations disrupting the HEN-1/Jeb ligand, SOC-1/DOS/GAB adaptor protein and SMA-5/ERK5 atypical MAP Kinase caused Scd phenotypes similar to that of mutant SCD-2. This group regulated expression from a TGF-beta-responsive GFP reporter. Here we find that a strain harboring a mutation in the uncharacterized SCD-4 is mutant for MLK-1, the C. elegans ortholog of mammalian Mixed Lineage Kinase and Drosophila slipper (slpr), a MAP3 kinase. We validated this finding by showing that a previously characterized deletion in MLK-1 caused a Scd phenotype similar to that of mutant SCD-4 and altered expression from the TGF-beta-responsive GFP reporter, suggesting that SCD-4 and MLK-1 are the same protein. Based on shared phenotypes and molecular identities, we hypothesize that MLK-1 functions as a MAP3K in the SCD-2/ALK cascade that signals through SMA-5/ERK5 MAP Kinase to modulate the output of the TGF-beta cascade controlling dauer formation in response to environmental cues.
ABSTRACT
Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.
Subject(s)
Complement Factor H/metabolism , Heparitin Sulfate/metabolism , Retina/metabolism , Animals , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , Cilia/metabolism , Complement Factor H/physiology , Heparitin Sulfate/physiology , Macular Degeneration/metabolism , Macular Degeneration/physiopathology , Neurons/metabolism , Transcription Factors/metabolismABSTRACT
BACKGROUND AND OBJECTIVES: Next generation sequencing (NGS) has promising applications in transfusion medicine. Exome sequencing (ES) is increasingly used in the clinical setting, and blood group interpretation is an additional value that could be extracted from existing data sets. We provide the first release of an open-source software tailored for this purpose and describe its validation with three blood group systems. MATERIALS AND METHODS: The DTM-Tools algorithm was designed and used to analyse 1018 ES NGS files from the ClinSeq® cohort. Predictions were correlated with serology for 5 antigens in a subset of 108 blood samples. Discrepancies were investigated with alternative phenotyping and genotyping methods, including a long-read NGS platform. RESULTS: Of 116 genomic variants queried, those corresponding to 18 known KEL, FY and JK alleles were identified in this cohort. 596 additional exonic variants were identified KEL, ACKR1 and SLC14A1, including 58 predicted frameshifts. Software predictions were validated by serology in 108 participants; one case in the FY blood group and three cases in the JK blood group were discrepant. Investigation revealed that these discrepancies resulted from (1) clerical error, (2) serologic failure to detect weak antigenic expression and (3) a frameshift variant absent in blood group databases. CONCLUSION: DTM-Tools can be employed for rapid Kell, Duffy and Kidd blood group antigen prediction from existing ES data sets; for discrepancies detected in the validation data set, software predictions proved accurate. DTM-Tools is open-source and in continuous development.
Subject(s)
Alleles , Blood Group Antigens/analysis , Blood Group Antigens/genetics , Exome Sequencing/methods , High-Throughput Nucleotide Sequencing/methods , Software , Duffy Blood-Group System/genetics , Genetic Variation , Genotyping Techniques , Humans , Membrane Glycoproteins/genetics , Membrane Transport Proteins/genetics , Metalloendopeptidases/genetics , Receptors, Cell Surface/genetics , Urea TransportersABSTRACT
SARS-CoV-2 infection ranges from asymptomatic to severe with lingering symptomatology in some. This prompted investigation of whether or not asymptomatic disease results in measurable immune activation post-infection. Immune activation following asymptomatic SARS-CoV-2 infection was characterized through a comparative investigation of the immune cell transcriptomes from 43 asymptomatic seropositive and 52 highly exposed seronegative individuals from the same community 4-6 weeks following a superspreading event. Few of the 95 individuals had underlying health issues. One seropositive individual reported Cystic Fibrosis and one individual reported Incontinentia pigmenti. No evidence of immune activation was found in asymptomatic seropositive individuals with the exception of the Cystic Fibrosis patient. There were no statistically significant differences in immune transcriptomes between asymptomatic seropositive and highly exposed seronegative individuals. Four positive controls, mildly symptomatic seropositive individuals whose blood was examined 3 weeks following infection, showed immune activation. Negative controls were four seronegative individuals from neighboring communities without COVID-19. All individuals remained in their usual state of health through a five-month follow-up after sample collection. In summary, whole blood transcriptomes identified individual immune profiles within a community population and showed that asymptomatic infection within a super-spreading event was not associated with enduring immunological activation.
Subject(s)
COVID-19/immunology , SARS-CoV-2/immunology , Transcriptome/immunology , Adaptive Immunity/genetics , Adolescent , Adult , Aged , Antibodies, Viral/blood , Antibodies, Viral/isolation & purification , Asymptomatic Infections , Austria , COVID-19/blood , COVID-19/diagnosis , COVID-19/transmission , COVID-19 Serological Testing/statistics & numerical data , Child , Child, Preschool , Contact Tracing/statistics & numerical data , Family Characteristics , Female , Follow-Up Studies , Host Microbial Interactions/genetics , Host Microbial Interactions/immunology , Humans , Immunity, Innate/genetics , Infant , Male , Middle Aged , RNA-Seq/statistics & numerical data , SARS-CoV-2/isolation & purification , Young AdultABSTRACT
Given their copy number differences and unique modes of inheritance, the evolved gene content and expression of sex chromosomes is unusual. In many organisms the X and Y chromosomes are inactivated in spermatocytes, possibly as a defense mechanism against insertions into unpaired chromatin. In addition to current sex chromosomes, Drosophila has a small gene-poor X-chromosome relic (4th) that re-acquired autosomal status. Here we use single cell RNA-Seq on fly larvae to demonstrate that the single X and pair of 4th chromosomes are specifically inactivated in primary spermatocytes, based on measuring all genes or a set of broadly expressed genes in testis we identified. In contrast, genes on the single Y chromosome become maximally active in primary spermatocytes. Reduced X transcript levels are due to failed activation of RNA-Polymerase-II by phosphorylation of Serine 2 and 5.
Subject(s)
Drosophila/genetics , Sex Chromosomes/genetics , Spermatocytes/metabolism , Animals , Drosophila/growth & development , Gene Expression Regulation , Genes, X-Linked/genetics , Genes, Y-Linked/genetics , Larva/genetics , Larva/growth & development , Male , Organ Specificity , RNA Polymerase II/metabolism , Sex Chromosomes/metabolism , Spermatogenesis/genetics , Testis/cytology , Testis/metabolism , Transcription, GeneticABSTRACT
Ciliary microtubules are subject to post-translational modifications that act as a "Tubulin Code" to regulate motor traffic, binding proteins and stability. In humans, loss of CCP1, a cytosolic carboxypeptidase and tubulin deglutamylating enzyme, causes infantile-onset neurodegeneration. In C. elegans, mutations in ccpp-1, the homolog of CCP1, result in progressive degeneration of neuronal cilia and loss of neuronal function. To identify genes that regulate microtubule glutamylation and ciliary integrity, we performed a forward genetic screen for suppressors of ciliary degeneration in ccpp-1 mutants. We isolated the ttll-5(my38) suppressor, a mutation in a tubulin tyrosine ligase-like glutamylase gene. We show that mutation in the ttll-4, ttll-5, or ttll-11 gene suppressed the hyperglutamylation-induced loss of ciliary dye filling and kinesin-2 mislocalization in ccpp-1 cilia. We also identified the nekl-4(my31) suppressor, an allele affecting the NIMA (Never in Mitosis A)-related kinase NEKL-4/NEK10. In humans, NEK10 mutation causes bronchiectasis, an airway and mucociliary transport disorder caused by defective motile cilia. C. elegans NEKL-4 localizes to the ciliary base but does not localize to cilia, suggesting an indirect role in ciliary processes. This work defines a pathway in which glutamylation, a component of the Tubulin Code, is written by TTLL-4, TTLL-5, and TTLL-11; is erased by CCPP-1; is read by ciliary kinesins; and its downstream effects are modulated by NEKL-4 activity. Identification of regulators of microtubule glutamylation in diverse cellular contexts is important to the development of effective therapies for disorders characterized by changes in microtubule glutamylation. By identifying C. elegans genes important for neuronal and ciliary stability, our work may inform research into the roles of the tubulin code in human ciliopathies and neurodegenerative diseases.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Carboxypeptidases/genetics , Nerve Degeneration/genetics , Peptide Synthases/genetics , Tubulin/genetics , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans/growth & development , Carrier Proteins/genetics , Cilia/genetics , Cilia/metabolism , Glutamic Acid/metabolism , Humans , Kinesins/genetics , Microtubules/genetics , Mutation/genetics , NIMA-Related Kinases/genetics , Nerve Degeneration/pathology , Neurons/metabolism , Neurons/pathology , Protein Processing, Post-Translational/geneticsABSTRACT
To investigate prevalence of ongoing activation of inflammation following asymptomatic SARS-CoV-2 infection we characterized immune cell transcriptomes from 43 asymptomatic seropositive and 52 highly exposed seronegative individuals with few underlying health issues following a community superspreading event. Four mildly symptomatic seropositive individuals examined three weeks after infection as positive controls demonstrated immunological activation. Approximately four to six weeks following the event, the two asymptomatic groups showed no significant differences. Two seropositive patients with underlying genetic disease impacting immunological activation were included (Cystic Fibrosis (CF), Nuclear factor-kappa B Essential Modulator (NEMO) deficiency). CF, but not NEMO, associated with significant immune transcriptome differences including some associated with severe SARS-CoV-2 infection (IL1B, IL17A, respective receptors). All subjects remained in their usual state of health from event through five-month follow-up. Here, asymptomatic infection resolved without evidence of prolonged immunological activation. Inclusion of subjects with underlying genetic disease illustrated the pathophysiological importance of context on impact of immunological response.
ABSTRACT
To investigate prevalence of ongoing activation of inflammation following asymptomatic SARS-CoV-2 infection we characterized immune cell transcriptomes from 43 asymptomatic seropositive and 52 highly exposed seronegative individuals with few underlying health issues following a community superspreading event. Four mildly symptomatic seropositive individuals examined three weeks after infection as positive controls demonstrated immunological activation. Approximately four to six weeks following the event, the two asymptomatic groups showed no significant differences. Two seropositive patients with underlying genetic disease impacting immunological activation were included (Cystic Fibrosis (CF), Nuclear factor-kappa B Essential Modulator (NEMO) deficiency). CF, but not NEMO, associated with significant immune transcriptome differences including some associated with severe SARS-CoV-2 infection (IL1B, IL17A, respective receptors). All subjects remained in their usual state of health from event through five-month follow-up. Here, asymptomatic infection resolved without evidence of prolonged immunological activation. Inclusion of subjects with underlying genetic disease illustrated the pathophysiological importance of context on impact of immunological response.
ABSTRACT
Topoisomerase II is an enzyme with important roles in chromosome biology. This enzyme relieves supercoiling and DNA and RNA entanglements generated during mitosis. Recent studies have demonstrated that Topoisomerase II is also involved in the segregation of homologous chromosomes during the first meiotic division. However, the function and regulation of Topoisomerase II in meiosis has not been fully elucidated. Here, we conducted a genetic suppressor screen in Caenorhabditis elegans to identify putative genes that interact with topoisomerase II during meiosis. Using a temperature-sensitive allele of topoisomerase II, top-2(it7ts), we identified eleven suppressors of top-2-induced embryonic lethality. We used whole-genome sequencing and a combination of RNAi and CRISPR/Cas9 genome editing to identify and validate the responsible suppressor mutations. We found both recessive and dominant suppressing mutations that include one intragenic and 10 extragenic loci. The extragenic suppressors consist of a known Topoisomerase II-interacting protein and two novel interactors. We anticipate that further analysis of these suppressing mutations will provide new insights into the function of Topoisomerase II during meiosis.