Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.
- MeSH
- buněčné jádro metabolismus MeSH
- Caenorhabditis elegans genetika metabolismus MeSH
- DNA metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- jaderné proteiny genetika metabolismus MeSH
- oprava DNA fyziologie MeSH
- poly-ADP-ribosylace MeSH
- polyadenosindifosfátribosa metabolismus MeSH
- posttranslační úpravy proteinů MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- zárodečné buňky MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Fanconi Anemia is a rare genetic disease associated with DNA repair defects, congenital abnormalities and infertility. Most of FA pathway is evolutionary conserved, allowing dissection and mechanistic studies in simpler model systems such as Caenorhabditis elegans. In the present study, we employed C. elegans to better understand the role of FA group D2 (FANCD2) protein in vivo, a key player in promoting genome stability. We report that localization of FCD-2/FANCD2 is dynamic during meiotic prophase I and requires its heterodimeric partner FNCI-1/FANCI. Strikingly, we found that FCD-2 recruitment depends on SPO-11-induced double-strand breaks (DSBs) but not RAD-51-mediated strand invasion. Furthermore, exposure to DNA damage-inducing agents boosts FCD-2 recruitment on the chromatin. Finally, analysis of genetic interaction between FCD-2 and BRC-1 (the C. elegans orthologue of mammalian BRCA1) supports a role for these proteins in different DSB repair pathways. Collectively, we showed a direct involvement of FCD-2 at DSBs and speculate on its function in driving meiotic DNA repair.
- MeSH
- Caenorhabditis elegans genetika růst a vývoj metabolismus MeSH
- dvouřetězcové zlomy DNA * MeSH
- meióza * MeSH
- oprava DNA * MeSH
- protein FANCD2 genetika metabolismus MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- rekombinace genetická * MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Parasitic nematodes transition between dramatically different free-living and parasitic stages, with correctly timed development and migration crucial to successful completion of their lifecycle. However little is known of the mechanisms controlling these transitions. microRNAs (miRNAs) negatively regulate gene expression post-transcriptionally and regulate development of diverse organisms. Here we used microarrays to determine the expression profile of miRNAs through development and in gut tissue of the pathogenic nematode Haemonchus contortus. Two miRNAs, mir-228 and mir-235, were enriched in infective L3 larvae, an arrested stage analogous to Caenorhabditis elegans dauer larvae. We hypothesized that these miRNAs may suppress development and maintain arrest. Consistent with this, inhibitors of these miRNAs promoted H. contortus development from L3 to L4 stage, while genetic deletion of C. elegans homologous miRNAs reduced dauer arrest. Epistasis studies with C. elegans daf-2 mutants showed that mir-228 and mir-235 synergise with FOXO transcription factor DAF-16 in the insulin signaling pathway. Target prediction suggests that these miRNAs suppress metabolic and transcription factor activity required for development. Our results provide novel insight into the expression and functions of specific miRNAs in regulating nematode development and identify miRNAs and their target genes as potential therapeutic targets to limit parasite survival within the host.
- MeSH
- Caenorhabditis elegans genetika MeSH
- cholesteny farmakologie MeSH
- delece genu MeSH
- druhová specificita MeSH
- genová ontologie MeSH
- Haemonchus účinky léků genetika růst a vývoj MeSH
- larva MeSH
- messenger RNA genetika metabolismus MeSH
- mikro RNA biosyntéza genetika MeSH
- proteiny Caenorhabditis elegans genetika MeSH
- receptor inzulinu genetika MeSH
- RNA helmintů biosyntéza genetika MeSH
- vývojová regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Cytokinins are phytohormones that are involved in many processes in plants, including growth, differentiation and leaf senescence. However, they also have various activities in animals. For example, kinetin and trans-zeatin can reduce levels of several aging markers in human fibroblasts. Kinetin can also protect mice against oxidative and glyoxidative stress, and prolong fruit flies' lifespan. Additionally, several cytokinins are currently used in cosmetics. To extend knowledge of the breadth of cytokinins' activities, we examined effects of natural cytokinin bases on the model nematode Caenorhabditis elegans. We found that kinetin, para-topolin and meta-topolin prolonged the lifespan of C. elegans. Kinetin also protected the organism against oxidative and heat stress. Furthermore, our results suggest that presence of reactive oxygen species, but not DAF-16 (the main effector of the insulin/insulin-like growth factor signaling pathway), is required for the beneficial effects of kinetin. Ultra-high performance liquid chromatography-tandem mass spectrometric analysis showed that kinetin is unlikely to occur naturally in C. elegans, but the worm efficiently absorbs and metabolizes it into kinetin riboside and kinetin riboside-5'-monophosphate.
- MeSH
- Caenorhabditis elegans účinky léků genetika fyziologie MeSH
- cytokininy farmakokinetika farmakologie MeSH
- dlouhověkost účinky léků fyziologie MeSH
- forkhead transkripční faktory genetika metabolismus MeSH
- inzulin metabolismus MeSH
- kinetin farmakokinetika farmakologie MeSH
- mutace MeSH
- oxidační stres účinky léků MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- reakce na tepelný šok účinky léků MeSH
- reaktivní formy kyslíku metabolismus MeSH
- regulátory růstu rostlin farmakokinetika farmakologie MeSH
- signální transdukce účinky léků MeSH
- termotolerance účinky léků MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Van Gogh-like (Vangl) and Prickle (Pk) are core components of the non-canonical Wnt planar cell polarity pathway that controls epithelial polarity and cell migration. Studies in vertebrate model systems have suggested that Vangl and Pk may also inhibit signaling through the canonical Wnt/β-catenin pathway, but the functional significance of this potential cross-talk is unclear. In the nematode C. elegans, the Q neuroblasts and their descendants migrate in opposite directions along the anteroposterior body axis. The direction of these migrations is specified by Wnt signaling, with activation of canonical Wnt signaling driving posterior migration, and non-canonical Wnt signaling anterior migration. Here, we show that the Vangl ortholog VANG-1 influences the Wnt signaling response of the Q neuroblasts by negatively regulating canonical Wnt signaling. This inhibitory activity depends on a carboxy-terminal PDZ binding motif in VANG-1 and the Dishevelled ortholog MIG-5, but is independent of the Pk ortholog PRKL-1. Moreover, using Vangl1 and Vangl2 double mutant cells, we show that a similar mechanism acts in mammalian cells. We conclude that cross-talk between VANG-1/Vangl and the canonical Wnt pathway is an evolutionarily conserved mechanism that ensures robust specification of Wnt signaling responses.
- MeSH
- beta-katenin genetika metabolismus MeSH
- buněčný rodokmen MeSH
- Caenorhabditis elegans cytologie genetika metabolismus MeSH
- fosfoproteiny genetika metabolismus MeSH
- geneticky modifikovaná zvířata MeSH
- geny helmintů MeSH
- homeodoménové proteiny genetika metabolismus MeSH
- intracelulární signální peptidy a proteiny genetika metabolismus MeSH
- mutace MeSH
- nervové kmenové buňky cytologie metabolismus MeSH
- polarita buněk genetika fyziologie MeSH
- protein dishevelled genetika metabolismus MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- rozvržení tělního plánu genetika fyziologie MeSH
- signální dráha Wnt genetika fyziologie MeSH
- transkripční faktory genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
One of the most studied phosphoinositides is phosphatidylinositol 4,5-bisphosphate (PIP2), which localizes to the plasma membrane, nuclear speckles, small foci in the nucleoplasm, and to the nucleolus in mammalian cells. Here, we show that PIP2 also localizes to the nucleus in prophase I, during the gametogenesis of C. elegans hermaphrodite. The depletion of PIP2 by type I PIP kinase (PPK-1) kinase RNA interference results in an altered chromosome structure and leads to various defects during meiotic progression. We observed a decreased brood size and aneuploidy in progeny, defects in synapsis, and crossover formation. The altered chromosome structure is reflected in the increased transcription activity of a tightly regulated process in prophase I. To elucidate the involvement of PIP2 in the processes during the C. elegans development, we identified the PIP2-binding partners, leucine-rich repeat (LRR-1) protein and proteasome subunit beta 4 (PBS-4), pointing to its involvement in the ubiquitin⁻proteasome pathway.
- MeSH
- buněčné jádro metabolismus MeSH
- Caenorhabditis elegans genetika růst a vývoj metabolismus MeSH
- chromozomy chemie MeSH
- fosfatidylinositol-4,5-difosfát metabolismus MeSH
- fosfotransferasy s alkoholovou skupinou jako akceptorem genetika MeSH
- gametogeneze * MeSH
- hermafroditické organismy genetika růst a vývoj metabolismus MeSH
- profáze meiózy I MeSH
- proteasomový endopeptidasový komplex metabolismus MeSH
- proteiny Caenorhabditis elegans genetika MeSH
- proteiny metabolismus MeSH
- RNA interference MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
ALKB-8 is a 2-oxoglutarate-dependent dioxygenase homologous to bacterial AlkB, which oxidatively demethylates DNA substrates. The mammalian AlkB family contains AlkB homologues denominated ALKBH1 to 8 and FTO. The C. elegans genome includes five AlkB-related genes, homologues of ALKBH1, 4, 6, 7, and 8, but lacks homologues of ALKBH2, 3, and 5 and FTO. ALKBH8 orthologues differ from other AlkB family members by possessing an additional methyltransferase module and an RNA binding N-terminal module. The ALKBH8 methyltransferase domain generates the wobble nucleoside 5-methoxycarbonylmethyluridine from its precursor 5-carboxymethyluridine and its (R)- and (S)-5-methoxycarbonylhydroxymethyluridine hydroxylated forms in tRNA Arg/UCG and tRNA Gly/UCC. The ALKBH8/ALKB-8 methyltransferase domain is highly similar to yeast TRM9, which selectively modulates translation of mRNAs enriched with AGA and GAA codons under both normal and stress conditions. In this report, we studied the role of alkb-8 in C. elegans. We show that downregulation of alkb-8 increases detection of lysosome-related organelles visualized by Nile red in vivo. Reversely, forced expression of alkb-8 strongly decreases the detection of this compartment. In addition, overexpression of alkb-8 applied in a pulse during the L1 larval stage increases the C. elegans lifespan.
- MeSH
- Caenorhabditis elegans embryologie enzymologie genetika MeSH
- dioxygenasy metabolismus MeSH
- dlouhověkost MeSH
- down regulace genetika MeSH
- embryo nesavčí metabolismus MeSH
- geneticky modifikovaná zvířata MeSH
- kyseliny ketoglutarové metabolismus MeSH
- larva metabolismus MeSH
- lyzozomy metabolismus MeSH
- methyltransferasy metabolismus MeSH
- operon MeSH
- promotorové oblasti (genetika) MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- RNA interference MeSH
- S-adenosylmethionin metabolismus MeSH
- stárnutí metabolismus MeSH
- vývojová regulace genové exprese MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5' end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5'→3' polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs.
- MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- intermediární filamenta genetika metabolismus MeSH
- jednovláknová DNA genetika MeSH
- multiproteinové komplexy metabolismus MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- rekombinační oprava DNA MeSH
- rekombinasa Rad51 genetika metabolismus MeSH
- transportní proteiny genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
The prorenin receptor (ATP6AP2) is a multifunctional transmembrane protein; it is a constituent of proton-translocating V-ATPase, a non-proteolytic activator of renin and an adaptor in the Wnt/β-catenin pathway. Here, we studied vha-20, one of the two prorenin receptor homologues that are identified by sequence similarity in the C. elegans genome. We show that vha-20 (R03E1.2) is prominently expressed in the intestine, in the excretory cell and in amphid neurons, tissues critical for regulation of ion and water management. The expression of vha-20 in the intestine is dependent on NHR-31, a nuclear receptor related to HNF4. VHA-20 is indispensable for normal larval development, acidification of the intestine, and is required for nutrient uptake. Inhibition of vha-20 by RNAi leads to complex deterioration of water and pH gradients at the level of the whole organism including distention of pseudocoelome cavity. This suggests new roles of prorenin receptor in the regulation of body ion and water management and in acidification of intestinal lumen in nematodes.
- MeSH
- Caenorhabditis elegans fyziologie MeSH
- homeostáza fyziologie MeSH
- koncentrace vodíkových iontů MeSH
- larva fyziologie MeSH
- polymerázová řetězová reakce MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- stanovení celkové genové exprese MeSH
- transkriptom MeSH
- vakuolární protonové ATPasy genetika metabolismus MeSH
- voda metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.
- MeSH
- Caenorhabditis elegans metabolismus MeSH
- DNA vazebné proteiny genetika metabolismus MeSH
- HEK293 buňky MeSH
- homologní rekombinace * MeSH
- jednovláknová DNA metabolismus MeSH
- komplex proteinů jaderného póru metabolismus MeSH
- lidé MeSH
- mutace MeSH
- proteiny Caenorhabditis elegans genetika metabolismus MeSH
- rekombinasa Rad51 metabolismus MeSH
- Saccharomyces cerevisiae - proteiny metabolismus MeSH
- Saccharomyces cerevisiae metabolismus MeSH
- transportní proteiny metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH