ABSTRACT
Cytotoxicity of cisplatin and mitomycin C (MMC) is ascribed largely to their ability to generate interstrand crosslinks (ICLs) in DNA, which block the progression of replication forks. The processing of ICLs requires the Fanconi anemia (FA) pathway, excision repair, and translesion DNA synthesis (TLS). It also requires homologous recombination (HR), which repairs double-strand breaks (DSBs) generated by cleavage of the blocked replication forks. Here we describe KIAA1018, an evolutionarily conserved protein that has an N-terminal ubiquitin-binding zinc finger (UBZ) and a C-terminal nuclease domain. KIAA1018 is a 5'-->3' exonuclease and a structure-specific endonuclease that preferentially incises 5' flaps. Like cells from FA patients, human cells depleted of KIAA1018 are sensitized to ICL-inducing agents and display chromosomal instability. The link of KIAA1018 to the FA pathway is further strengthened by its recruitment to DNA damage through interaction of its UBZ domain with monoubiquitylated FANCD2. We therefore propose to name KIAA1018 FANCD2-associated nuclease, FAN1.
Subject(s)
Cross-Linking Reagents/pharmacology , DNA Repair , Exodeoxyribonucleases/metabolism , Fanconi Anemia Complementation Group D2 Protein/metabolism , Mitomycin/pharmacology , Amino Acid Sequence , Animals , Caenorhabditis elegans , Cell Line , DNA Breaks, Double-Stranded , DNA Damage/drug effects , Endodeoxyribonucleases , Endonucleases/metabolism , Exodeoxyribonucleases/chemistry , Humans , Molecular Sequence Data , Multifunctional Enzymes , Phosphodiesterase I/metabolism , Protein Structure, Tertiary , Sequence AlignmentABSTRACT
Caspases are key components of apoptotic pathways. Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases. Here, we describe four conserved RNA-binding proteins (RBPs)-PUF-8, MEX-3, GLD-1, and CGH-1-that sequentially repress the CED-3 caspase in distinct regions of the Caenorhabditis elegans germline. We demonstrate that GLD-1 represses ced-3 mRNA translation via two binding sites in its 3' untranslated region (UTR), thereby ensuring a dual control of unwanted cell death: at the level of p53/CEP-1 and at the executioner caspase level. Moreover, we identified seven RBPs that regulate human caspase-3 expression and/or activation, including human PUF-8, GLD-1, and CGH-1 homologs PUM1, QKI, and DDX6. Given the presence of unusually long executioner caspase 3' UTRs in many metazoans, translational control of executioner caspases by RBPs might be a strategy used widely across the animal kingdom to control apoptosis.
Subject(s)
Apoptosis/genetics , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caspases/genetics , Caspases/metabolism , Gene Expression Regulation, Developmental , RNA-Binding Proteins/metabolism , 3' Untranslated Regions/genetics , Animals , Binding Sites , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Germ Cells/cytology , HeLa Cells , Humans , RNA Processing, Post-TranscriptionalABSTRACT
Synthesis of ribosomal RNA by RNA polymerase I (RNA pol I) is an elemental biological process and is key for cellular homeostasis. In a forward genetic screen in C. elegans designed to identify DNA damage-response factors, we isolated a point mutation of RNA pol I, rpoa-2(op259), that leads to altered rRNA synthesis and a concomitant resistance to ionizing radiation (IR)-induced germ cell apoptosis. This weak apoptotic IR response could be phenocopied when interfering with other factors of ribosome synthesis. Surprisingly, despite their resistance to DNA damage, rpoa-2(op259) mutants present a normal CEP-1/p53 response to IR and increased basal CEP-1 activity under normal growth conditions. In parallel, rpoa-2(op259) leads to reduced Ras/MAPK pathway activity, which is required for germ cell progression and physiological germ cell death. Ras/MAPK gain-of-function conditions could rescue the IR response defect in rpoa-2(op259), pointing to a function for Ras/MAPK in modulating DNA damage-induced apoptosis downstream of CEP-1. Our data demonstrate that a single point mutation in an RNA pol I subunit can interfere with multiple key signalling pathways. Ribosome synthesis and growth-factor signalling are perturbed in many cancer cells; such an interplay between basic cellular processes and signalling might be critical for how tumours evolve or respond to treatment.
Subject(s)
Apoptosis/radiation effects , Mitogen-Activated Protein Kinase Kinases/genetics , RNA, Ribosomal/biosynthesis , Ribosomes/genetics , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/genetics , DNA Damage/genetics , DNA Damage/radiation effects , Germ Cells/radiation effects , Humans , Mitogen-Activated Protein Kinase Kinases/metabolism , Point Mutation , RNA Polymerase I/genetics , RNA, Ribosomal/radiation effects , Radiation, Ionizing , Signal Transduction , Tumor Suppressor Protein p53/geneticsABSTRACT
Despite the many advantages of Caenorhabditis elegans, biochemical approaches to study tissue-specific gene expression in post-embryonic stages are challenging. Here, we report a novel experimental approach for efficient determination of tissue-specific transcriptomes involving the rapid release and purification of nuclei from major tissues of post-embryonic animals by fluorescence-activated nuclei sorting (FANS), followed by deep sequencing of linearly amplified 3'-end regions of transcripts (3'-end-seq). We employed these approaches to compile the transcriptome of the developed C. elegans intestine and used this to analyse tissue-specific cleavage and polyadenylation. In agreement with intestinal-specific gene expression, highly expressed genes have enriched GATA-elements in their promoter regions and their functional properties are associated with processes that are characteristic for the intestine. We systematically mapped pre-mRNA cleavage and polyadenylation sites, or polyA sites, including more than 3000 sites that have previously not been identified. The detailed analysis of the 3'-ends of the nuclear mRNA revealed widespread alternative polyA site use (APA) in intestinally expressed genes. Importantly, we found that intestinal polyA sites that undergo APA tend to have U-rich and/or A-rich upstream auxiliary elements that may contribute to the regulation of 3'-end formation in the intestine.
Subject(s)
Caenorhabditis elegans/genetics , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing , Polyadenylation , Sequence Analysis, RNA , Animals , Caenorhabditis elegans/metabolism , Cell Fractionation/methods , Cell Nucleus/genetics , Flow Cytometry , Intestinal Mucosa/metabolism , TranscriptomeABSTRACT
Background: DNA polymerase gamma (POLG)-related disorders are a group of rare neurodegenerative mitochondrial diseases caused by pathogenic variants in POLG, the gene encoding POLG. Patients may experience a range of signs and symptoms, including seizures, vision loss, myopathy, neuropathy, developmental impairment or regression, and liver failure. The diseases follow a progressive, degenerative course, with most affected individuals dying within 3 months-12 years of diagnosis. At present, there are no effective treatments for POLG-related disorders. Methods: In this study we report the interim 6-month data from a long term open-label, single arm phase 2 trial, in which we assessed the safety and efficacy of combination therapy with deoxycytidine and deoxythymidine (dC/dT) in children with POLG-related disorders. dC/dT was given enterally in powder form, dissolved in water. The primary outcome measures included Newcastle Mitochondrial Disease Scale (NMDS) score, serum growth differentiation factor 15 (GDF-15; a biomarker of mitochondrial dysfunction), electroencephalography (EEG), seizure diary, and blood and urine tests to assess end organ and mitochondrial function. Secondary outcome measures included recording of all adverse events to evaluate the safety of the intervention. The trial is registered with ClinicalTrials.gov, NCT04802707 (https://clinicaltrials.gov/ct2/show/NCT04802707). Data were collected from 14 October, 2021 to 13 December, 2023. Findings: We present 6-month interim data from the first ten people with POLG-related disorders enrolled in the trial, six with Alpers-Huttenlocher syndrome, two with ataxia-neuropathy spectrum, and two who do not fit into a classical POLG-related phenotype. During the 6 months of treatment, NMDS score improved from a mean of 27.3 at baseline to 20.7 at 6 months (estimated difference 6.0; 95% CI 2.5-∞). GDF-15 values remained stable or decreased in all patients; the mean decreased from 1031 pg/ml to 729 pg/ml (estimated difference 200; 95% CI 12-∞). 8/10 patients had abnormal baseline EEG; improvement in EEG was seen in 5 of these 8. There were no significant changes in other blood and urine testing. Regarding adverse events, two patients experienced diarrhea that spontaneously resolved. Interpretation: dC/dT is a promising treatment option for people with POLG-related disorders. Further research is needed to assess the long-term safety and efficacy in POLG-related disorders, as well as safety and efficacy in other mitochondrial DNA depletion disorders. Funding: This study was primarily funded by the Liam Foundation, with additional funding from the Savoy Foundation, Grand Défi Pierre Lavoie Foundation, and Fonds de Recherche du Québec - Santé.
ABSTRACT
The germ line of the nematode C. elegans provides a paradigm to study essential developmental concepts like stem cell differentiation and apoptosis. Here, we have created a computational model encompassing these developmental landmarks and the resulting movement of germ cells along the gonadal tube. We have used a technique based on molecular dynamics (MD) to model the physical movement of cells solely based on the force that arises from dividing cells. This novel way of using MD to drive the model enables calibration of simulation and experimental time. Based on this calibration, the analysis of our model shows that it is in accordance with experimental observations. In addition, the model provides insights into kinetics of molecular pathways within individual cells as well as into physical aspects like the cell density along the germ line and in local neighbourhoods of individual germ cells. In the future, the presented model can be used to test hypotheses about diverse aspects of development like stem cell division or programmed cell death. An iterative process of evolving this model and experimental testing in the model system C. elegans will provide new insights into key developmental aspects.
Subject(s)
Apoptosis/physiology , Caenorhabditis elegans/physiology , Cell Differentiation/physiology , Cell Movement/physiology , Models, Biological , Animals , Caenorhabditis elegans/cytology , Cell Count , Cell Lineage , Computer Simulation , Female , Germ Cells/cytology , Kinetics , Male , Stem Cells/cytologyABSTRACT
Corticosteroids are commonly used in children with bacterial meningitis; however, there are very few data regarding possible utility in neonates, particularly those born premature. We describe our experience using hydrocortisone in the treatment of a girl born at 26 weeks, 6 days gestation. She had suffered profound brain injury following late onset group B streptococcus sepsis and meningitis, and developed drug-resistant seizures. Because seizures continued despite treatment with phenobarbital, phenytoin, levetiracetam, lacosamide, and midazolam, intravenous hydrocortisone was added. We observed a marked decrease in focal electrographic seizures within 2 days of initiation of hydrocortisone. This experience suggests that corticosteroids could be a treatment option for drug-resistant seizures and status epilepticus in preterm neonates, particularly those with bacterial meningitis.
ABSTRACT
BACKGROUND: Lung volume reduction aims to improve symptoms by reducing hyperinflation. Endoscopic approaches so far have generally been hampered in their efficacy by collateral ventilation (CV). We sought to determine the safety and feasibility of a new endoscopic lung volume reduction approach independent of the effects of CV. METHODS: Patients with severe emphysema were eligible. Inclusion and exclusion criteria were modeled after the National Emphysema Treatment Trial (NETT) study. Homogenous and heterogeneous disease was allowed. Treatment consisted of the placement of coils into the parenchyma of the most diseased area with the intent of achieving parenchymal compression. Primary endpoints were safety and feasibility assessments. Secondary endpoints were efficacy outcomes. RESULTS: Eleven patients underwent 21 procedures. Procedures were performed under general anesthesia and lasted 45+/-15 minutes and per procedure 4.9+/-0.6 coils were placed. All procedures were well tolerated. The total follow-up time was 7-11 months and in that time 33 adverse events were reported, none of them severe. No pneumothorax occurred. Efficacy seemed better in heterogeneous rather than homogenous disease. CONCLUSION: Endoscopic lung volume reduction with coils is safe and feasible. Further studies of the efficacy are indicated.
Subject(s)
Bronchoscopy/methods , Emphysema/therapy , Pneumonectomy/methods , Aged , Bronchoscopy/adverse effects , Emphysema/physiopathology , Female , Follow-Up Studies , Humans , Male , Middle Aged , Pilot Projects , Pneumonectomy/adverse effects , Severity of Illness Index , Time Factors , Treatment OutcomeABSTRACT
The occurrence of blood-borne prion transmission incidents calls for identification of potential prion carriers. However, current methods for intravital diagnosis of prion disease rely on invasive tissue biopsies and are unsuitable for large-scale screening. Sensitive biomarkers may help meeting this need. Here we scanned the genome for transcripts elevated upon prion infection and encoding secreted proteins. We found that alpha(1)-antichymotrypsin (alpha(1)-ACT) was highly upregulated in brains of scrapie-infected mice. Furthermore, alpha(1)-ACT levels were dramatically increased in urine of patients suffering from sporadic Creutzfeldt-Jakob disease, and increased progressively throughout the disease. Increased alpha(1)-ACT excretion was also found in cases of natural prion disease of animals. Therefore measurement of urinary alpha(1)-ACT levels may be useful for monitoring the efficacy of therapeutic regimens for prion disease, and possibly also for deferring blood and organ donors that may be at risk of transmitting prion infections.