ABSTRACT
MicroRNA-mediated gene silencing is a fundamental mechanism in the regulation of gene expression. It remains unclear how the efficiency of RNA silencing could be influenced by RNA-binding proteins associated with the microRNA-induced silencing complex (miRISC). Here we report that fused in sarcoma (FUS), an RNA-binding protein linked to neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), interacts with the core miRISC component AGO2 and is required for optimal microRNA-mediated gene silencing. FUS promotes gene silencing by binding to microRNA and mRNA targets, as illustrated by its action on miR-200c and its target ZEB1. A truncated mutant form of FUS that leads its carriers to an aggressive form of ALS, R495X, impairs microRNA-mediated gene silencing. The C. elegans homolog fust-1 also shares a conserved role in regulating the microRNA pathway. Collectively, our results suggest a role for FUS in regulating the activity of microRNA-mediated silencing.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Gene Silencing , MicroRNAs/metabolism , RNA, Helminth/metabolism , RNA-Binding Protein FUS/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Animals , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , HEK293 Cells , Humans , Mice , MicroRNAs/genetics , RNA, Helminth/genetics , RNA-Binding Protein FUS/geneticsABSTRACT
Genetic diversity is maintained by continuing generation and removal of variants. While examining over 800,000 DNA variants in wild isolates of Caenorhabditis elegans, we made a discovery that the proportions of variant types are not constant across the C. elegans genome. The variant proportion is defined as the fraction of a specific variant type (e.g. single nucleotide polymorphism (SNP) or indel) within a broader set of variants (e.g. all variants or all non-SNPs). The proportions of most variant types show a correlation with the recombination rate. These correlations can be explained as a result of a concerted action of two mutation mechanisms, which we named Morgan and Sanger mechanisms. The two proposed mechanisms act according to the distinct components of the recombination rate, specifically the genetic and physical distance. Regression analysis was used to explore the characteristics and contributions of the two mutation mechanisms. According to our model, ~20-40% of all mutations in C. elegans wild populations are derived from programmed meiotic double strand breaks, which precede chromosomal crossovers and thus may be the point of origin for the Morgan mechanism. A substantial part of the known correlation between the recombination rate and variant distribution appears to be caused by the mutations generated by the Morgan mechanism. Mathematically integrating the mutation model with background selection model gives a more complete depiction of how the variant landscape is shaped in C. elegans. Similar analysis should be possible in other species by examining the correlation between the recombination rate and variant landscape within the context of our mutation model.
Subject(s)
Caenorhabditis elegans/genetics , Genetics, Population , Models, Genetic , Mutation/genetics , Polymorphism, Single Nucleotide/genetics , Selection, Genetic/genetics , Animals , Chromosome Mapping , Computer Simulation , Genome/genetics , Models, Statistical , Recombination, Genetic/geneticsABSTRACT
Transactive response DNA-binding protein (TARDBP/TDP-43), a heterogeneous nuclear ribonucleoprotein (hnRNP) with diverse activities, is a common denominator in several neurodegenerative disorders, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Orthologs of TDP-43 exist in animals ranging from mammals to invertebrates. Here, we systematically studied mutant Caenorhabditis elegans lacking the nematode TDP-43 ortholog, TDP-1. Heterologous expression of human TDP-43 rescued the defects in C. elegans lacking TDP-1, suggesting their functions are conserved. Although the tdp-1 mutants exhibited deficits in fertility, growth, and locomotion, loss of tdp-1 attenuated defects in several C. elegans models of proteotoxicity. Loss of tdp-1 suppressed defects in transgenic C. elegans expressing TDP-43 or CuZn superoxide dismutase, both of which are associated with proteotoxicity in neurodegenerative diseases. Loss of tdp-1 also reduced defects in mutant animals lacking the heat shock factor HSF-1. Transcriptional profiling demonstrated that the loss of TDP-1 altered expression of genes functioning in RNA processing and protein folding. Furthermore, the absence of tdp-1 extended the life span in C. elegans. The life span extension required a FOXO transcriptional factor DAF-16 but not HSF-1. These results suggest that the C. elegans TDP-1 has a role in the regulation of protein homeostasis and aging.
Subject(s)
Caenorhabditis elegans/metabolism , DNA-Binding Proteins/biosynthesis , Homeostasis/physiology , Longevity/physiology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , DNA-Binding Proteins/genetics , Forkhead Transcription Factors , Frontotemporal Lobar Degeneration/genetics , Frontotemporal Lobar Degeneration/metabolism , Genetic Complementation Test , Humans , Mutation , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Greater molecular divergence and genetic diversity are present in regions of high recombination in many species. Studies describing the correlation between variant abundance and recombination rate have long focused on recombination in the context of linked selection models, whereby interference between linked sites under positive or negative selection reduces genetic diversity in regions of low recombination. Here, we show that indels, especially those of intermediate sizes, are enriched relative to single nucleotide polymorphisms in regions of high recombination in C. elegans. To explain this phenomenon, we reintroduce an alternative model that emphasizes the mutagenic effect of recombination. To extend the analysis, we examine the variants with a phylogenetic context and discuss how different models could be examined together. The number of variants generated by recombination in natural populations could be substantial including possibly the majority of some indel subtypes. Our work highlights the potential importance of a mutagenic effect of recombination, which could have a significant role in the shaping of natural genetic diversity.
Subject(s)
Caenorhabditis elegans , Recombination, Genetic , Animals , Caenorhabditis elegans/genetics , Phylogeny , Selection, Genetic , Polymorphism, Single Nucleotide , Genetic VariationABSTRACT
Cell lines are useful tools to facilitate in vitro studies of many biological and molecular processes. We describe a new permanent fibroblast-type cell line obtained from disaggregated Xenopus tropicalis limb bud. The cell line population doubling time was ~24 h. Its karyotype was genetically stable with a chromosome number of 2n = 21 and a chromosome 10 trisomy. These cells could be readily transfected and expressed transgenes faithfully. We obtained stable transformants using transposon-based gene transfer technology. These cells responded to thyroid hormone and thus can provide a complementary research tool to study thyroid hormone signaling events. In conclusion, this cell line baptized "Speedy" should prove useful to couple in vitro and in vivo biological studies in the X. tropicalis frog model.
Subject(s)
Cell Line , Xenopus/genetics , Animals , DNA Transposable Elements , Gene Order , Gene Transfer Techniques , Genes, Reporter , Genetic Vectors/genetics , Karyotype , Primary Cell Culture , Signal Transduction/drug effects , Thyroid Hormones/pharmacology , Transfection , Transgenes , Xenopus/metabolismABSTRACT
Genetic mapping is used in forward genetics to narrow the list of candidate mutations and genes corresponding to the mutant phenotype of interest. Even with modern advances in biology such as efficient identification of candidate mutations by whole-genome sequencing, mapping remains critical in pinpointing the responsible mutation. Here we describe a simple, fast, and affordable mapping toolkit that is particularly suitable for mapping in Caenorhabditis elegans. This mapping method uses insertion-deletion polymorphisms or indels that could be easily detected instead of single nucleotide polymorphisms in commonly used Hawaiian CB4856 mapping strain. The materials and methods were optimized so that mapping could be performed using tiny amount of genetic material without growing many large populations of mutants for DNA purification. We performed mapping of previously known and unknown mutations to show strengths and weaknesses of this method and to present examples of completed mapping. For situations where Hawaiian CB4856 is unsuitable, we provide an annotated list of indels as a basis for fast and easy mapping using other wild isolates. Finally, we provide rationale for using this mapping method over other alternatives as a part of a comprehensive strategy also involving whole-genome sequencing and other methods.
Subject(s)
Caenorhabditis elegans , Chromosome Mapping , INDEL Mutation , Animals , PhenotypeABSTRACT
Thermotolerance of an organism is a complex trait that is influenced by a multitude of genetic and environmental factors. Many factors controlling thermotolerance in Caenorhabditis elegans are known to extend life. To understand the regulation of thermotolerance, we performed a genetic screen for mutants with better survival at warm temperature. Here we identified by dauer survival a tax-2 mutation and several mutations disrupting an insulin signaling pathway including the daf-2 gene. While the tax-2 mutant has improved thermotolerance and long life span, the newly identified daf-2 and other insulin signaling mutants, unlike the canonical daf-2(e1370), do not show improved thermotolerance despite being long-lived. Examination of tax-2 mutations and their mutant phenotypes suggest that the control of thermotolerance is not coupled with the control of life span or dauer survival. With genetic interaction studies, we concluded that tax-2 has complex roles in life span and dauer survival and that tax-2 is a negative regulator of thermotolerance independent of other known thermotolerance genes including those in the insulin signaling pathway. Moreover, cold growth temperature during development weakens the improved thermotolerance associated with tax-2 and other thermotolerance-inducing mutations. Together, this study reveals previously unknown genetic and environmental factors controlling thermotolerance and their complex relationship with life span regulation.
ABSTRACT
A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Both loss-of-function and gain-of-function mechanisms have been proposed to underlie this disease, but the pathogenic pathways are not fully understood. To better understand the involvement of different cell types in the pathogenesis of ALS, we systematically analyzed the distribution of promoter activity of the mouse ortholog of C9orf72 in the central nervous system. We demonstrate that C9orf72 promoter activity is widespread in both excitatory and inhibitory neurons as well as in oligodendrocytes and oligodendrocyte precursor cells. In contrast, few microglia and astrocytes exhibit detectable C9orf72 promoter activity. Although at a gross level, the distribution of C9orf72 promoter activity largely follows overall cellular density, we found that it is selectively enriched in subsets of neurons and glial cells that degenerate in ALS. Specifically, we show that C9orf72 promoter activity is enriched in corticospinal and spinal motor neurons as well as in oligodendrocytes in brain regions that are affected in ALS. These results suggest that cell autonomous changes in both neurons and glia may contribute to C9orf72-mediated disease, as has been shown for mutations in superoxide dismutase-1 (SOD1).
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , C9orf72 Protein/genetics , Central Nervous System/pathology , Amyotrophic Lateral Sclerosis/pathology , Animals , Central Nervous System/cytology , Lac Operon , Mice , Mice, Knockout , Motor Neurons , Oligodendroglia , Promoter Regions, GeneticABSTRACT
The development of the Caenorhabditis elegans vulva requires the involution of epithelial cells and provides a model for organ morphogenesis. Mutations in C. elegans sqv (squashed vulva) genes affect both vulval morphogenesis and embryonic development. We found that sqv-4 encodes a protein similar to UDP-glucose dehydrogenases and showed that the SQV-4 protein specifically catalyzes the conversion of UDP-glucose to UDP-glucuronic acid, which is essential for the biosynthesis of chondroitin and heparan sulfate proteoglycans. SQV-4 is expressed in the vulva and in oocytes, among many other cells, and SQV-4 levels are dramatically increased in a specific subset of vulval cells during vulval morphogenesis. We propose that the regulation of UDP-glucuronic acid production in a specific subset of vulval cells helps determine the shape of the vulva.
Subject(s)
Caenorhabditis elegans Proteins/genetics , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Genes, Helminth/physiology , Uridine Diphosphate Glucose Dehydrogenase/genetics , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/embryology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Cell Count , DNA, Complementary , Female , Helminth Proteins/genetics , Helminth Proteins/metabolism , Homeodomain Proteins/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Morphogenesis , Receptors, Notch , Sequence Homology, Amino Acid , Uridine Diphosphate Glucose/metabolism , Uridine Diphosphate Glucose Dehydrogenase/metabolism , Uridine Diphosphate Glucuronic Acid/metabolism , Vulva/embryologyABSTRACT
Recent findings indicate that glycosaminoglycans can play important roles in animal development. The genes sqv-3, -7, and -8, which are necessary for vulval morphogenesis in Caenorhabditis elegans, affect the biosynthesis of chondroitin and heparan sulfate glycosaminoglycans. We cloned sqv-1 and showed that the SQV-1 protein is a type II transmembrane protein that functions as a UDP-glucuronic acid decarboxylase. SQV-1 localizes to punctate cytoplasmic compartments and colocalizes with the SQV-7 nucleotide-sugar transporter, which probably acts in the Golgi apparatus. SQV-1 and SQV-7 are both expressed in the vulva and in oocytes, where they likely act in vulval morphogenesis and embryonic development, respectively. Progeny of sqv-7 and sqv-1 null mutants fail to initiate cytokinesis, possibly because they are unable to separate the plasma membrane from the eggshell, a defect analogous to that of incomplete vulval invagination.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Carboxy-Lyases/metabolism , Carrier Proteins/metabolism , Golgi Apparatus/metabolism , Membrane Proteins/metabolism , Monosaccharide Transport Proteins , Nucleobase, Nucleoside, Nucleotide, and Nucleic Acid Transport Proteins , Amino Acid Sequence , Animals , Animals, Genetically Modified , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/genetics , Carbohydrate Metabolism , Carboxy-Lyases/genetics , Cytoplasm/enzymology , Female , Humans , Membrane Proteins/genetics , Molecular Sequence Data , Morphogenesis , Nucleotides/metabolism , Rabbits , Staining and Labeling/methods , Uridine Diphosphate Glucuronic Acid/metabolism , Uridine Diphosphate Xylose/metabolismABSTRACT
Defects in glycosaminoglycan biosynthesis disrupt animal development and can cause human disease. So far much of the focus on glycosaminoglycans has been on heparan sulphate. Mutations in eight squashed vulva (sqv) genes in Caenorhabditis elegans cause defects in cytokinesis during embryogenesis and in vulval morphogenesis during postembryonic development. Seven of the eight sqv genes have been shown to control the biosynthesis of the glycosaminoglycans chondroitin and heparan sulphate. Here we present the molecular identification and characterization of the eighth gene, sqv-5. This gene encodes a bifunctional glycosyltransferase that is probably localized to the Golgi apparatus and is responsible for the biosynthesis of chondroitin but not heparan sulphate. Our findings show that chondroitin is crucial for both cytokinesis and morphogenesis during C. elegans development.
Subject(s)
Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Chondroitin/biosynthesis , Morphogenesis , Vulva/embryology , Vulva/metabolism , Alleles , Amino Acid Sequence , Animals , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/analysis , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/genetics , Caenorhabditis elegans Proteins/metabolism , Cloning, Molecular , Female , Gene Deletion , Gene Expression Profiling , Glycosyltransferases/analysis , Glycosyltransferases/chemistry , Glycosyltransferases/genetics , Glycosyltransferases/metabolism , Heparitin Sulfate/biosynthesis , Molecular Sequence Data , Sequence Alignment , Substrate Specificity , Vulva/enzymologyABSTRACT
In mutants defective in any of eight Caenorhabditis elegans sqv (squashed vulva) genes, the vulval extracellular space fails to expand during vulval morphogenesis. Strong sqv mutations result in maternal-effect lethality, caused in part by the failure of the progeny of homozygous mutants to initiate cytokinesis and associated with the failure to form an extracellular space between the egg and the eggshell. Recent studies have implicated glycosaminoglycans in these processes. Here we report the cloning and characterization of sqv-2 and sqv-6. sqv-6 encodes a protein similar to human xylosyltransferases. Transfection of sqv-6 restored xylosyltransferase activity to and rescued the glycosaminoglycan biosynthesis defect of a xylosyltransferase mutant hamster cell line. sqv-2 encodes a protein similar to human galactosyltransferase II. A recombinant SQV-2 fusion protein had galactosyltransferase II activity with substrate specificity similar to that of human galactosyltransferase II. We conclude that C. elegans SQV-6 and SQV-2 likely act in concert with other SQV proteins to catalyze the stepwise formation of the proteoglycan core protein linkage tetrasaccharide GlcAbeta1,3Galbeta1, 3Galbeta1,4Xylbeta-O-(Ser), which is common to the two major types of glycosaminoglycans in vertebrates, chondroitin and heparan sulfate. Our results strongly support a model in which C. elegans vulval morphogenesis and zygotic cytokinesis depend on the expression of glycosaminoglycans.