A Werner syndrome protein homolog affects C. elegans development, growth rate, life span and sensitivity to DNA damage by acting at a DNA damage checkpoint

A Werner syndrome protein homolog in C. elegans (WRN-1) was immunolocalized to the nuclei of germ cells, embryonic cells, and many other cells of larval and adult worms. When wrn-1 expression was inhibited by RNA interference (RNAi), a slight reduction in C. elegans life span was observed, with accompanying signs of premature aging, such as earlier accumulation of lipofuscin and tissue deterioration in the head. In addition, various developmental defects, including small, dumpy, ruptured, transparent body, growth arrest and bag of worms, were induced by RNAi. The frequency of these defects was accentuated by gamma-irradiation, implying that they were derived from spontaneous or induced DNA damage. wrn-1(RNAi) worms showed accelerated larval growth irrespective of gamma-irradiation, and pre-meiotic germ cells had an abnormal checkpoint response to DNA replication blockage. These observations suggest that WRN-1 acts as a checkpoint protein for DNA damage and replication blockage. This idea is also supported by an accelerated S phase in wrn-1(RNAi) embryonic cells. wrn-1(RNAi) phenotypes similar to those of Werner syndrome, such as premature aging and short stature, suggest wrn-1-deficient C. elegans as a useful model organism for Werner syndrome.     

Deficiency of the Caenorhabditis elegans DNA polymerase eta homologue increases sensitivity to UV radiation during germ-line development

Defects in the human XPV/POLH gene result in the variant form of the disease xeroderma pigmentosum (XP-V). The gene encodes DNA polymerase eta (Poleta), which catalyzes translesion synthesis (TLS) past UV-induced cyclobutane pyrimidine dimers (CPDs) and other lesions. To further understand the roles of Poleta in multicellular organisms, we analyzed phenotypes caused by suppression of Caenorhabditis elegans POLH (Ce-POLH) by RNA interference (RNAi). F1 and F2 progeny from worms treated by Ce-POLH-specific RNAi grew normally, but F1 eggs laid by worms treated by RNAi against Ce-POLD, which encodes Poldelta did not hatch. These results suggest that Poldelta but not Poleta is essential for C. elegans embryogenesis. Poleta-targeted embryos UV-irradiated after egg laying were only moderately sensitive. In contrast, Poleta-targeted embryos UV-irradiated prior to egg laying exhibited severe sensitivity, indicating that Poleta contributes significantly to damage tolerance in C. elegans in early embryogenesis but only modestly at later stages. As early embryogenesis is characterized by high levels of DNA replication, Poleta may confer UV resistance in C. elegans, perhaps by catalyzing TLS in early embryogenesis.     

Nematode chondroitin polymerizing factor showing cell-/organ-specific expression is indispensable for chondroitin synthesis and embryonic cell division

Chondroitin polymerization was first demonstrated in vitro when human chondroitin synthase (ChSy) was coexpressed with human chondroitin polymerizing factor (ChPF), which is homologous to ChSy but has little glycosyltransferase activity. To analyze the biological function of chondroitin, the Caenorhabditis elegans ortholog of human ChSy (sqv-5) was recently cloned, and the expression of its product was depleted by RNA-mediated interference (RNAi) and deletion mutagenesis. Blocking of chondroitin synthesis resulted in defects of cytokinesis in early embryogenesis, and eventually, cell division stopped. Here, we cloned the ortholog of human ChPF in C. elegans, PAR2.4. Despite little glycosyltransferase activity of the gene product, chondroitin polymerization was demonstrated as in the case of mammals when PAR2.4 was coexpressed with cChSy in vitro. The worm phenotypes including the reversion of cytokinesis, observed after the depletion of PAR2.4 by RNAi, were very similar to the cChSy (sqv-5)-RNAi phenotypes. Thus, PAR2.4 in addition to cChSy is indispensable for the biosynthesis of chondroitin in C. elegans, and the two cooperate to synthesize chondroitin in vivo. The expression of the PAR2.4 protein was observed in seam cells, which can act as neural stem cells in early embryonic lineages. The expression was also detected in vulva and distal tip cells of the growing gonad arms from L3 through to the young adult stage. These findings are consistent with the notion that chondroitin is involved in the organogenesis of the vulva and maturation of the gonad and also indicative of an involvement in distal tip cell migration and neural development.     

The maintenance of neuromuscular function requires UBC-25 in Caenorhabditis elegans

Caenorhabditis elegans gene ubc-25 encodes a novel type of an E2 ubiquitin transferase domain (UBCc) protein, which is highly conserved in multicellular animals, but which is not present in the genomes of fungi or plants. To identify the cellular localization of UBC-25 during the development of C. elegans, we used a ubc-25::gfp reporter gene construct. These experiments showed that ubc-25 expression starts during embryogenesis and that it is restricted to neurons and muscle cells in all later stages of development as well as in adult animals. RNA interference with ubc-25 caused late-onset paralysis of most muscular functions such as locomotion, egg laying, and defecation. We therefore propose that ubc-25 in C. elegans is required for the maintenance (homeostasis) of neuromuscular functions by contributing to a tissue specific protein modification pathway, and we speculate that the adult onset phenotype results from the accumulation of target proteins which fail to be degraded.     

Inducible systemic RNA silencing in Caenorhabditis elegans

Introduction of double-stranded RNA (dsRNA) can elicit a gene-specific RNA interference response in a variety of organisms and cell types. In many cases, this response has a systemic character in that silencing of gene expression is observed in cells distal from the site of dsRNA delivery. The molecular mechanisms underlying the mobile nature of RNA silencing are unknown. For example, although cellular entry of dsRNA is possible, cellular exit of dsRNA from normal animal cells has not been directly observed. We provide evidence that transgenic strains of Caenorhabditis elegans transcribing dsRNA from a tissue-specific promoter do not exhibit comprehensive systemic RNA interference phenotypes. In these same animals, modifications of environmental conditions can result in more robust systemic RNA silencing. Additionally, we find that genetic mutations can influence the systemic character of RNA silencing in C. elegans and can separate mechanisms underlying systemic RNA silencing into tissue-specific components. These data suggest that trafficking of RNA silencing signals in C. elegans is regulated by specific physiological and genetic factors.     

Production of antisense RNA leads to effective and specific inhibition of gene expression in C. elegans muscle.

We have used an antisense strategy to effectively disrupt the expression of two genes encoding myofilament proteins present in C. elegans body wall muscles. DNA segments from the unc-22 and unc-54 genes have been placed in reverse orientation in vectors designed to produce RNA in body wall muscles. When the resulting plasmids are injected into oocytes, progeny with defects in muscle function are produced. These animals have phenotypes consistent with reduction and/or elimination of function of the gene to which antisense RNA has been produced: twitching and disorganization of muscle filaments for the unc-22 antisense constructs and lack of muscle tone, slow movement, and egg laying defects for the unc-54 antisense constructs. A fraction of the affected animals transmit the defective-muscle trait to subsequent generations. In these cases the transforming DNA is present at high copy number and cosegregates with the observed muscle defects. We have examined several of the unc-22 antisense plasmid transformed lines to determine the mechanistic basis for the observed phenotypes. The RNA product of the endogenous unc-22 locus is present at normal levels and this RNA is properly spliced in the region homologous to the antisense RNA. No evidence for modification of this RNA by deamination of adenosine to inosine was found. In affected animals the level of protein product from the endogenous unc-22 locus is greatly reduced. Antisense RNA produced from the transforming DNA was detected and was much more abundant than 'sense' RNA from the endogenous locus. These data suggest that the observed phenotypes result from interference with a late step in gene expression, such as transport into the cytoplasm or translation.     

Loss of RecQ5 leads to spontaneous mitotic defects and chromosomal aberrations in Drosophila melanogaster

RecQ5 belongs to the RecQ DNA helicase family that includes genes causative of Bloom, Werner, and Rothmund-Thomson syndromes. Although no human disease has been genetically linked to a mutation in RecQ5, Drosophila melanogaster RecQ5 is highly expressed in early embryos, suggesting an important role for it in the DNA metabolism of the early embryo. In this present study, we generated RecQ5 mutants in D. melanogaster. Embryos lacking maternally derived RecQ5 contained irregular nuclei in early embryogenesis. These irregular nuclei emerged in nuclear cycle 11–13, lost cell-cycle markers, and were located below the surface monolayer of nuclei. By time-lapse microscopy, these irregular nuclei were observed not to divide, whereas all neighboring nuclei proceeded through normal mitotic division with synchrony. These data suggest that the irregular nuclei exited from the nuclear division cycle. This phenotype is reminiscent of the effect of X-ray irradiation on wild-type embryos and was rescued by expression of RecQ5. Thus, the maternal supply of RecQ5 is important for the nuclear cycles in syncytical embryos. Furthermore, the frequencies of spontaneous and induced chromosomal aberrations were increased in RecQ5 mutant neuroblasts. These data imply that DNA damage accumulates spontaneously in RecQ5 mutants. Therefore, endogenous genomic damage may be produced in Drosophila development, and RecQ5 would be involved in the maintenance of genomic stability by suppressing the accumulation of DNA damage.     

Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion

SEC-23 is a component of coat protein complex II (COPII)-coated vesicles involved in the endoplasmic reticulum-to-Golgi transport pathway of eukaryotes. During postembryonic life, Caenorhabditis elegans is surrounded by a collagenous exoskeleton termed the cuticle. From a screen for mutants defective in cuticle secretion, we identified and characterized a sec-23 mutant of C. elegans. By sequence homology, C. elegans has only the single sec-23 gene described herein. In addition to the cuticle secretion defect, mutants fail to complete embryonic morphogenesis. However, they progress through the earlier stages of embryogenesis, including gastrulation, and achieve substantial morphogenesis before death. We demonstrated a maternal component of SEC-23 function sufficient for progression through the earlier stages of embryogenesis and explaining the limited phenotype of the zygotic mutant. By RNA-mediated interference, we investigated the effects of perturbing COPII function during various postembryonic stages. During larval stages, major defects in cuticle synthesis and molting were observed. In the adult hermaphrodite, reduction of SEC-23 function by RNA-mediated interference caused a rapid onset of sterility, with defects in oogenesis including early maturation of the germline nuclei, probably a result of the observed loss of the GLP-1 receptor from the membrane surfaces adjacent to the developing germline nuclei.     

Eukaryotic translation initiation factor 5B activity regulates larval growth rate and germline development in Caenorhabditis elegans

In C. elegans, a population of proliferating germ cells is maintained via GLP-1/Notch signaling; in the absence of GLP-1 signaling, germ cells prematurely enter meiosis and differentiate. We previously identified ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we report that iffb-1 (initiation factor five B) is an ego gene. iffb-1 encodes the sole C. elegans isoform of eukaryotic translation initiation factor 5B, a protein essential for translation. We have used RNA interference and a deletion mutation to determine the developmental consequences of reduced iffb-1 activity. Our data indicate that maternal iffb-1 gene expression is sufficient for embryogenesis, and zygotic iffb-1 expression is required for development beyond late L1/early L2 stage. Partial reduction in iffb-1 expression delays larval development and can severely disrupt proliferation and differentiation of germ cells. We hypothesize that germline development is particularly sensitive to iffb-1 expression level.     

Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes

Caenorhabditis elegans has a single lamin gene, designated lmn-1 (previously termed CeLam-1). Antibodies raised against the lmn-1 product (Ce-lamin) detected a 64-kDa nuclear envelope protein. Ce-lamin was detected in the nuclear periphery of all cells except sperm and was found in the nuclear interior in embryonic cells and in a fraction of adult cells. Reductions in the amount of Ce-lamin protein produce embryonic lethality. Although the majority of affected embryos survive to produce several hundred nuclei, defects can be detected as early as the first nuclear divisions. Abnormalities include rapid changes in nuclear morphology during interphase, loss of chromosomes, unequal separation of chromosomes into daughter nuclei, abnormal condensation of chromatin, an increase in DNA content, and abnormal distribution of nuclear pore complexes (NPCs). Under conditions of incomplete RNA interference, a fraction of embryos escaped embryonic arrest and continue to develop through larval life. These animals exhibit additional phenotypes including sterility and defective segregation of chromosomes in germ cells. Our observations show that lmn-1 is an essential gene in C. elegans, and that the nuclear lamins are involved in chromatin organization, cell cycle progression, chromosome segregation, and correct spacing of NPCs.     

Drosophila RecQ5 is involved in proper progression of early spermatogenesis

RecQ5, a member of the conserved RecQ DNA helicase family, is required for the maintenance of genome stability. The human RECQL5 gene is expressed ubiquitously in almost all tissues, with strong expression in the testes (Shimamoto et al., 2000). However, it remains to be elucidated in which cells RecQ5 is expressed and how RecQ5 functions in the testes. In this present study we analyzed the expression of RecQ5 in Drosophila testes. The RecQ5 protein was specifically expressed in germline cells in larval, pupal, and adult testes. Drosophila RecQ5 was localized in nuclei of male germline stem cells, spermatogoniablasts, spermatogonia, and early spermatocytes. As growth of the early spermatocyte proceeded, the amount of RecQ5 increased in the nuclei. However, before maturation of the spermatocyte, the level of RecQ5 declined. Thus, RecQ5 expression was regulated. Furthermore, we compared recq5 mutant testes with the wild-type ones. The most conspicuous alterations were swelling of the apical region of and an increase in the number of spermatocytes in the recq5 testis, suggesting a relative accumulation of spermatocytes in the recq5 mutant testes. Therefore, Drosophila RecQ5 may contribute to the proper progression from germline stem cells to spermatocytes for maintenance of genome stability.     

Biochemical characterization of the WRN-1 RecQ helicase of Caenorhabditis elegans

The highly conserved RecQ helicases are essential for the maintenance of genomic stability. Werner syndrome protein, WRN, is one of five human RecQ helicase homologues, and a deficiency of the protein causes a hereditary premature aging disorder that is characterized by genomic instability. A WRN orthologue, wrn-1 lacking the exonuclease domain, has been identified in the nematode Caenorhabditis elegans. wrn-1(RNAi) in C. elegans has a shortened life span, increased sensitivity to DNA damage, and accelerated aging phenotypes. However, little is known about its enzymatic activity. We purified the recombinant C. elegans WRN-1 protein (CeWRN-1) and then investigated its substrate specificity in vitro to improve our understanding of its function in vivo. We found that CeWRN-1 is an ATP-dependent 3'-5' helicase capable of unwinding a variety of DNA structures such as forked duplexes, Holliday junctions, bubble substrates, D-loops, and flap duplexes, and 3'-tailed duplex substrates. Distinctly, CeWRN-1 is able to unwind a long forked duplex compared to human WRN. Furthermore, CeWRN-1 helicase activity on a long DNA duplex is stimulated by C. elegans replication protein A (CeRPA) that is shown to interact with CeWRN-1 by a dot blot. The ability of CeWRN-1 to unwind these DNA structures may improve the access for DNA repair and replication proteins that are important for preventing the accumulation of abnormal structures, contributing to genomic stability.     

The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans

The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity.     

The near demise and subsequent revival of classical genetics for investigating Caenorhabditis elegans embryogenesis: RNAi meets next-generation DNA sequencing

Molecular genetic investigation of the early Caenorhabditis elegans embryo has contributed substantially to the discovery and general understanding of the genes, pathways, and mechanisms that regulate and execute developmental and cell biological processes. Initially, worm geneticists relied exclusively on a classical genetics approach, isolating mutants with interesting phenotypes after mutagenesis and then determining the identity of the affected genes. Subsequently, the discovery of RNA interference (RNAi) led to a much greater reliance on a reverse genetics approach: reducing the function of known genes with RNAi and then observing the phenotypic consequences. Now the advent of next-generation DNA sequencing technologies and the ensuing ease and affordability of whole-genome sequencing are reviving the use of classical genetics to investigate early C. elegans embryogenesis.