Cytosolic aconitase and ferritin are regulated by iron in Caenorhabditis elegans

Iron regulatory protein-1 (IRP-1) is a cytosolic RNA-binding protein that is a regulator of iron homeostasis in mammalian cells. IRP-1 binds to RNA structures, known as iron-responsive elements, located in the untranslated regions of specific mRNAs, and it regulates the translation or stability of these mRNAs. Iron regulates IRP-1 activity by converting it from an RNA-binding apoprotein into a [4Fe-4S] cluster protein exhibiting aconitase activity. IRP-1 is widely found in prokaryotes and eukaryotes. Here, we report the biochemical characterization and regulation of an IRP-1 homolog in Caenorhabditis elegans (GEI-22/ACO-1). GEI-22/ACO-1 is expressed in the cytosol of cells of the hypodermis and the intestine. Like mammalian IRP-1/aconitases, GEI-22/ACO-1 exhibits aconitase activity and is post-translationally regulated by iron. Although GEI-22/ACO-1 shares striking resemblance to mammalian IRP-1, it fails to bind RNA. This is consistent with the lack of iron-responsive elements in the C. elegans ferritin genes, ftn-1 and ftn-2. While mammalian ferritin H and L mRNAs are translationally regulated by iron, the amounts of C. elegans ftn-1 and ftn-2 mRNAs are increased by iron and decreased by iron chelation. Excess iron did not significantly alter worm development but did shorten their life span. These studies indicated that iron homeostasis in C. elegans shares some similarities with those of vertebrates.     

Caenorhabditis elegans body mechanics are regulated by body wall muscle tone

Body mechanics in the nematode Caenorhabditis elegans are central to both mechanosensation and locomotion. Previous work revealed that the mechanics of the outer shell, rather than internal hydrostatic pressure, dominates stiffness. This shell is comprised of the cuticle and the body wall muscles, either of which could contribute to the body mechanics. Here, we tested the hypothesis that the muscles are an important contributor by modulating muscle tone using optogenetic and pharmacological tools, and measuring animal stiffness using piezoresistive microcantilevers. As a proxy for muscle tone, we measured changes in animal length under the same treatments. We found that treatments that induce muscle contraction generally resulted in body shortening and stiffening. Conversely, methods to relax the muscles more modestly increased length and decreased stiffness. The results support the idea that body wall muscle activation contributes significantly to and can modulate C. elegans body mechanics. Modulation of body stiffness would enable nematodes to tune locomotion or swimming gaits and may have implications in touch sensation.     

Sperm development and motility are regulated by PP1 phosphatases in Caenorhabditis elegans

Sperm from different species have evolved distinctive motility structures, including tubulin-based flagella in mammals and major sperm protein (MSP)-based pseudopods in nematodes. Despite such divergence, we show that sperm-specific PP1 phosphatases, which are required for male fertility in mouse, function in multiple processes in the development and motility of Caenorhabditis elegans amoeboid sperm. We used live-imaging analysis to show the PP1 phosphatases GSP-3 and GSP-4 (GSP-3/4) are required to partition chromosomes during sperm meiosis. Postmeiosis, tracking fluorescently labeled sperm revealed that both male and hermaphrodite sperm lacking GSP-3/4 are immotile. Genetic and in vitro activation assays show lack of GSP-3/4 causes defects in pseudopod development and the rate of pseudopodial treadmilling. Further, GSP-3/4 are required for the localization dynamics of MSP. GSP-3/4 shift localization in concert with MSP from fibrous bodies that sequester MSP at the base of the pseudopod, where directed MSP disassembly facilitates pseudopod contraction. Consistent with a role for GSP-3/4 as a spatial regulator of MSP disassembly, MSP is mislocalized in sperm lacking GSP-3/4. Although a requirement for PP1 phosphatases in nematode and mammalian sperm suggests evolutionary conservation, we show PP1s have independently evolved sperm-specific paralogs in separate lineages. Thus PP1 phosphatases are highly adaptable and employed across a broad range of sexually reproducing species to regulate male fertility.     

Crossover Heterogeneity in the Absence of Hotspots in Caenorhabditis elegans

Crossovers play mechanical roles in meiotic chromosome segregation, generate genetic diversity by producing new allelic combinations, and facilitate evolution by decoupling linked alleles. In almost every species studied to date, crossover distributions are dramatically nonuniform, differing among sexes and across genomes, with spatial variation in crossover rates on scales from whole chromosomes to subkilobase hotspots. To understand the regulatory forces dictating these heterogeneous distributions a crucial first step is the fine-scale characterization of crossover distributions. Here we define the wild-type distribution of crossovers along a region of the C. elegans chromosome II at unprecedented resolution, using recombinant chromosomes of 243 hermaphrodites and 226 males. We find that well-characterized large-scale domains, with little fine-scale rate heterogeneity, dominate this region’s crossover landscape. Using the Gini coefficient as a summary statistic, we find that this region of the C. elegans genome has the least heterogeneous fine-scale crossover distribution yet observed among model organisms, and we show by simulation that the data are incompatible with a mammalian-type hotspot-rich landscape. The large-scale structural domains—the low-recombination center and the high-recombination arm—have a discrete boundary that we localize to a small region. This boundary coincides with the arm-center boundary defined both by nuclear-envelope attachment of DNA in somatic cells and GC content, consistent with proposals that these features of chromosome organization may be mechanical causes and evolutionary consequences of crossover recombination.     

Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.     

Maternal mRNAs are regulated by diverse P body-related mRNP granules during early Caenorhabditis elegans development

Processing bodies (P bodies) are conserved mRNA-protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body-like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3' untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development.     

The Synaptonemal Complex Shapes the Crossover Landscape Through Cooperative Assembly,Crossover Promotion and Crossover Inhibition During Caenorhabditis elegans Meiosis

The synaptonemal complex (SC) is a highly ordered proteinaceous structure that assembles at the interface between aligned homologous chromosomes during meiotic prophase. The SC has been demonstrated to function both in stabilization of homolog pairing and in promoting the formation of interhomolog crossovers (COs). How the SC provides these functions and whether it also plays a role in inhibiting CO formation has been a matter of debate. Here we provide new insight into assembly and function of the SC by investigating the consequences of reducing (but not eliminating) SYP-1, a major structural component of the SC central region, during meiosis in Caenorhabditis elegans. First, we find an increased incidence of double CO (DCO) meiotic products following partial depletion of SYP-1 by RNAi, indicating a role for SYP-1 in mechanisms that normally limit crossovers to one per homolog pair per meiosis. Second, syp-1 RNAi worms exhibit both a strong preference for COs to occur on the left half of the X chromosome and a significant bias for SYP-1 protein to be associated with the left half of the chromosome, implying that the SC functions locally in promoting COs. Distribution of SYP-1 on chromosomes in syp-1 RNAi germ cells provides strong corroboration for cooperative assembly of the SC central region and indicates that SYP-1 preferentially associates with X chromosomes when it is present in limiting quantities. Further, the observed biases in the distribution of both COs and SYP-1 protein support models in which synapsis initiates predominantly in the vicinity of pairing centers (PCs). However, discontinuities in SC structure and clear gaps between localized foci of PC-binding protein HIM-8 and X chromosome-associated SYP-1 stretches allow refinement of models for the role of PCs in promoting synapsis. Our data suggest that the CO landscape is shaped by a combination of three attributes of the SC central region: a CO-promoting activity that functions locally at CO sites, a cooperative assembly process that enables CO formation in regions distant from prominent sites of synapsis initiation, and CO-inhibitory role(s) that limit CO number.REDUCTION in ploidy during sexual reproduction depends on the ability to form pairwise associations between homologous chromosomes. The homolog pairing process typically culminates in an arrangement in which the homologs are aligned in parallel along their lengths, with a highly ordered proteinaceous structure known as the synaptonemal complex (SC) located at the interface between them. Further, in most organisms, pairwise associations between homologs are solidified through the formation of crossovers (COs) between their DNA molecules, a process that is completed within the context of the SC.The SC has long been recognized as a hallmark cytological feature of meiosis. It was discovered on the basis of its highly ordered structure and location at the interface between aligned chromosomes in electron microscopy images of nuclei at the pachytene stage of meiotic prophase (Moses 1956, 2006). Each of the homologs is associated with one of the two lateral elements (LEs) of the SC, which are composed of cohesin complexes and other meiosis-specific structural and regulatory proteins (reviewed in Mlynarczyk-Evans and Villeneuve 2010). The LEs are connected by a highly ordered latticework of transverse filaments, and often a pronounced central element, that comprise the central region of the SC. The protein components of the SC central region are very poorly conserved at the primary sequence level, but the major central region proteins identified from diverse species share in common extended regions of predicted coiled coil structure.The SC has been demonstrated to have at least two conserved functions in meiotic prophase. First, the SC serves to stabilize and maintain tight associations along the lengths of aligned homologs (reviewed in Mlynarczyk-Evans and Villeneuve 2010). This is true both in organisms in which SC assembly is coupled to formation of recombination intermediates (e.g., budding yeast, mouse, and Arabidopsis) and in organisms in which formation of SC between homologs can occur independently of recombination (e.g., Caenorhabditis elegans and Drosophila). Second, SC central region proteins play a role in promoting maturation of recombination intermediates into crossover products (reviewed in De Boer and Heyting 2006). How the SC functions to promote CO formation is not well understood. Moreover, whether the SC might also have additional functions that help to ensure a successful outcome of meiosis has been a matter of debate.In addition to its roles in stabilization of pairing and promoting CO formation, the SC has also been proposed to function in inhibiting CO formation (Egel 1978, 1995; Maguire 1988). This idea of the SC playing an inhibitory role in recombination dates almost as far back as the discovery of the SC itself. Finding a highly ordered structure with a zipper-like appearance extending along the length of each homolog pair naturally gave rise to speculation that it might play a role in the phenomenon of crossover interference, defined as the ability of a (nascent) CO to inhibit the formation of other COs nearby on the same chromosome pair (Muller 1916; Hillers 2004). It was variously proposed either that the SC might serve as a conduit of information along a chromosome pair (e.g., undergoing a distance-dependent “change in state” to inhibit COs) or that SC polymerization might itself confer CO inhibition (Egel 1978; Maguire 1988; Sym and Roeder 1994).Early analysis of the budding yeast mutants lacking Zip1, a major structural component of the SC central region, initially seemed to support the idea that the SC central region played a key role in CO interference, as zip1 mutants formed COs at 30–50% of wild-type levels and the residual COs did not display interference (Sym and Roeder 1994). However, these data were subsequently reinterpreted by postulating that the major interference-sensitive meiotic CO pathway is eliminated in the zip1 mutant and that the residual COs form by an alternative pathway that is not subject to interference (Zalevsky et al. 1999; de los Santos et al. 2003). According to this two-pathway view, the lack of interference in the zip1 mutant can be readily explained without invoking a role for Zip1 in the interference mechanism per se. Conversely, Page and Hawley found that Drosophila females expressing a mutant form of the fly SC central region protein C(3)G retained substantial interference between residual COs despite exhibiting incomplete synapsis, implying that complete SC formation was not required for CO interference (Page and Hawley 2001). In light of these and other findings (e.g., Borner et al. 2004; Fung et al. 2004), the idea that the SC might play a role in inhibiting CO formation fell from favor.In this study, we revisit a potential role for the SC central region in inhibiting CO formation, using the C. elegans experimental system. Several features make this an interesting system for investigating factors that promote and/or inhibit COs during meiosis. First, essentially all COs in C. elegans depend on conserved meiotic CO-promoting machinery (i.e., Msh4 and Msh5) and on SC central region proteins (SYP-1, -2, -3, and -4), so analysis is generally not complicated by residual COs forming by alternative pathways (Zalevsky et al. 1999; Kelly et al. 2000; MacQueen et al. 2002; Colaiacovo et al. 2003; Smolikov et al. 2007a, 2009). Second, C. elegans hermaphrodites exhibit robust CO control, with COs usually being limited to one per homolog pair per meiosis (Hillers and Villeneuve 2003; Nabeshima et al. 2004; Hammarlund et al. 2005). Consequently, circumstances that give rise to double crossover (DCO) meiotic products can be inferred to represent impairment of mechanisms that normally inhibit CO formation. Finally, COs are distributed nonuniformly along the lengths of the chromosomes, with each chromosome containing broad domains of relatively high CO frequency flanking a more central domain where CO frequency is low (Brenner 1974; Barnes et al. 1995; Rockman and Kruglyak 2009), providing an opportunity to evaluate how factors that promote and/or inhibit COs contribute to this landscape.Our strategy was to use RNAi to reduce the levels of wild-type SYP-1 protein without eliminating synapsis entirely and then to examine the effects on CO frequency and distribution. This approach indeed revealed a role for SC central region protein SYP-1 in mechanisms that normally limit the number of COs per homolog pair. Further, it also revealed a role for the SC central region in determining CO distribution, presumably by enabling formation of COs in chromosome regions distant from the dominant site of synapsis initiation. Finally, our experimental design also afforded us the opportunity to evaluate spatial distribution of the SC in the context of limiting amounts of a key central region component. This analysis provided additional insight into the process of SC assembly and the role of cis-acting meiotic pairing centers in this process.     

Crossover distribution and high interference for both the X chromosome and an autosome during oogenesis and spermatogenesis in Caenorhabditis elegans

Regulation of both the number and the location of crossovers during meiosis is important for normal chromosome segregation. We used sequence-tagged site polymorphisms to examine the distribution of all crossovers on the X chromosome during oogenesis and on one autosome during both oogenesis and spermatogenesis in Caenorhabditis elegans. The X chromosome has essentially one crossover during oogenesis, with only three possible double crossover exceptions among 220 recombinant X chromosomes. All three had one of the two crossovers in the same chromosomal interval, suggesting that crossovers in that interval do not cause interference. No other interval was associated with double crossovers. Very high interference was also found on an autosome during oogenesis, implying that each chromosome has only one crossover during oogenesis. During spermatogenesis, recombination on this autosome was reduced by approximately 30% compared to oogenesis, but the relative distribution of the residual crossovers was only slightly different. In contrast to previous results with other autosomes, no double crossover chromosomes were observed. Despite an increased frequency of nonrecombinant chromosomes, segregation of a nonrecombinant autosome during spermatogenesis appears to occur normally. This indicates that an achiasmate segregation system helps to ensure faithful disjunction of autosomes during spermatogenesis.     

Interplay between Structure-Specific Endonucleases for Crossover Control during Caenorhabditis elegans Meiosis

The number and distribution of crossover events are tightly regulated at prophase of meiosis I. The resolution of Holliday junctions by structure-specific endonucleases, including MUS-81, SLX-1, XPF-1 and GEN-1, is one of the main mechanisms proposed for crossover formation. However, how these nucleases coordinately resolve Holliday junctions is still unclear. Here we identify both the functional overlap and differences between these four nucleases regarding their roles in crossover formation and control in the Caenorhabditis elegans germline. We show that MUS-81, XPF-1 and SLX-1, but not GEN-1, can bind to HIM-18/SLX4, a key scaffold for nucleases. Analysis of synthetic mitotic defects revealed that MUS-81 and SLX-1, but not XPF-1 and GEN-1, have overlapping roles with the Bloom syndrome helicase ortholog, HIM-6, supporting their in vivo roles in processing recombination intermediates. Taking advantage of the ease of genetic analysis and high-resolution imaging afforded by C. elegans, we examined crossover designation, frequency, distribution and chromosomal morphology in single, double, triple and quadruple mutants of the structure-specific endonucleases. This revealed that XPF-1 functions redundantly with MUS-81 and SLX-1 in executing crossover formation during meiotic double-strand break repair. Analysis of crossover distribution revealed that SLX-1 is required for crossover suppression at the center region of the autosomes. Finally, analysis of chromosome morphology in oocytes at late meiosis I stages uncovered that SLX-1 and XPF-1 promote meiotic chromosomal stability by preventing formation of chromosomal abnormalities. We propose a model in which coordinate action between structure-specific nucleases at different chromosome domains, namely MUS-81, SLX-1 and XPF-1 at the arms and SLX-1 at the center region, exerts positive and negative regulatory roles, respectively, for crossover control during C. elegans meiosis.     

Synaptic polarity depends on phosphatidylinositol signaling regulated by myo-inositol monophosphatase in Caenorhabditis elegans

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP(2) on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP(2) synthesis through inhibition of IMPase. We propose that the PIP(2) signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity.     

Sequence comparisons of developmentally regulated collagen genes of Caenorhabditis elegans

Collagen genes col-6, col-7 (partial), col-8, col-14 and col-19 from the nematode Caenorhabditis elegans were sequenced, and compared to the previously sequenced genes col-1 and col-2. The genes are between 1.0 and 1.2 kb in length, and each includes one or two short introns. The presumptive promoter regions contain sequences similar to the eukaryotic TATA promoter element. Two distinct, conserved sequences were found in the presumptive promoter regions of, respectively, the dauer larva-specific genes col-2 and col-6, and the primarily adult-specific genes col-7 and col-19. The domain structures of the collagen polypeptides are similar: each polypeptide contains two triple-helix forming (Gly-X-Y)n domains, one of 30-33 amino acids (aa), and the other of 127-132 aa. The latter domain is interrupted by one to three short (2-8 aa) non-(Gly-X-Y)n segments that occur at relatively conserved locations in each polypeptide. Sets of cysteine residues flank the (Gly-X-Y)n domains in all of the polypeptides. The genes can be placed into three families based upon amino acid sequence similarities. Genes within a family do not always exhibit similar developmental expression programs, suggesting that structural and regulatory regions of the genes have evolved separately. The codon usage in the genes is highly asymmetrical, with adenine appearing in the third position of 85% of the glycine codons, and 93% of the proline codons.     

A Highlights from MBoC Selection: Mutations in Caenorhabditis elegans him-19 Show Meiotic Defects That Worsen with Age

From a screen for meiotic Caenorhabditis elegans mutants based on high incidence of males, we identified a novel gene, him-19, with multiple functions in prophase of meiosis I. Mutant him-19(jf6) animals show a reduction in pairing of homologous chromosomes and subsequent bivalent formation. Consistently, synaptonemal complex formation is spatially restricted and possibly involves nonhomologous chromosomes. Also, foci of the recombination protein RAD-51 occur delayed or cease altogether. Ultimately, mutation of him-19 leads to chromosome missegregation and reduced offspring viability. The observed defects suggest that HIM-19 is important for both homology recognition and formation of meiotic DNA double-strand breaks. It therefore seems to be engaged in an early meiotic event, resembling in this respect the regulator kinase CHK-2. Most astonishingly, him-19(jf6) hermaphrodites display worsening of phenotypes with increasing age, whereas defects are more severe in female than in male meiosis. This finding is consistent with depletion of a him-19-dependent factor during the production of oocytes. Further characterization of him-19 could contribute to our understanding of age-dependent meiotic defects in humans.     

Caenorhabditis elegans body wall muscles are simple actuators

Over the past four decades, one of the simplest nervous systems across the animal kingdom, that of the nematode worm Caenorhabditis elegans, has drawn increasing attention. This system is the subject of an intensive concerted effort to understand the behaviour of an entire living animal, from the bottom up and the top down. C. elegans locomotion, in particular, has been the subject of a number of models, but there is as yet no general agreement about the key (rhythm generating) elements. In this paper we investigate the role of one component of the locomotion subsystem, namely the body wall muscles, with a focus on the role of inter-muscular gap junctions. We construct a detailed electrophysiological model which suggests that these muscles function, to a first approximation, as mere actuators and have no obvious rhythm generating role. Furthermore, we show that within our model inter-muscular coupling is too weak to have a significant electrical effect. These results rule out muscles as key generators of locomotion, pointing instead to neural activity patterns. More specifically, the results imply that the reduced locomotion velocity observed in unc-9 mutants is likely to be due to reduced neuronal rather than inter-muscular coupling.     

5'-AMP-activated protein kinase signaling in Caenorhabditis elegans

5'-AMP-activated protein kinase (AMPK) has been called "the metabolic master switch" because of its central role in regulating fuel homeostasis. AMPK, a heterotrimeric serine/threonine protein kinase composed of alpha, beta, and gamma subunits, is activated by upstream kinases and by 5'-AMP in response to various nutritional and stress signals. Downstream effects include regulation of metabolism, protein synthesis, cell growth, and mediation of the actions of a number of hormones, including leptin. However, AMPK research represents a young and growing field; hence, there are many unanswered questions regarding the control and action of AMPK. This review presents evidence for the existence of AMPK signaling pathways in Caenorhabditis elegans, a genetically tractable model organism that has yet to be fully exploited to elucidate AMPK signaling mechanisms.     

Thymidylate synthase and dihydropyrimidine dehydrogenase levels are associated with response to 5-fluorouracil in Caenorhabditis elegans

5-Fluorouracil (5-FU), a pyrimidine antagonist, has a long history in cancer treatment. The targeted pyrimidine biosynthesis pathway includes dihydropyrimidine dehydrogenase (DPD), which converts 5-FU to an inactive metabolite, and thymidylate synthase (TS), which is a major target of 5-FU. Using Caenorhabditis elegans as a model system to study the functional and resistance mechanisms of anti-cancer drugs, we examined these two genes in order to determine the extent of molecular conservation between C. elegans and humans. Overexpression of the worm DPD and TS homologs (DPYD-1 and Y110A7A.4, respectively) suppressed germ cell death following 5-FU exposure. In addition, DPYD-1 depletion by RNAi resulted in 5-FU sensitivity, while treatment with Y110A7A.4 RNAi and 5-FU resulted in similar patterns of embryonic death. Thus, the pathway of 5-FU function appears to be highly conserved between C. elegans and humans at the molecular level.     

Genome integrity is regulated by the Caenorhabditis elegans Rad51D homolog rfs-1

Multiple mechanisms ensure genome maintenance through DNA damage repair, suppression of transposition, and telomere length regulation. The mortal germline (Mrt) mutants in Caenorhabditis elegans are defective in maintaining genome integrity, resulting in a progressive loss of fertility over many generations. Here I show that the high incidence of males (him)-15 locus, defined by the deficiency eDf25, is allelic to rfs-1, the sole rad-51 paralog group member in C. elegans. The rfs-1/eDf25 mutant displays a Mrt phenotype and mutant animals manifest features of chromosome fusions prior to the onset of sterility. Unlike other Mrt genes, rfs-1 manifests fluctuations in telomere lengths and functions independently of telomerase. These data suggest that rfs-1 is a novel regulator of genome stability.     

Analysis of the role of Caenorhabditis elegans GC-AG introns in regulated splicing

GC-AG introns represent 0.7% of total human pre-mRNA introns. To study the function of GC-AG introns in splicing regulation, 196 cDNA-confirmed GC-AG introns were identified in Caenorhabditis elegans. These represent 0.6% of the cDNA- confirmed intron data set for this organism. Eleven of these GC-AG introns are involved in alternative splicing. In a comparison of the genomic sequences of homologous genes between C.elegans and Caenorhabditis briggsae for 26 GC-AG introns, the C at the +2 position is conserved in only five of these introns. A system to experimentally test the function of GC-AG introns in alternative splicing was developed. Results from these experiments indicate that the conserved C at the +2 position of the tenth intron of the let-2 gene is essential for developmentally regulated alternative splicing. This C allows the splice donor to function as a very weak splice site that works in balance with an alternative GT splice donor. A weak GT splice donor can functionally replace the GC splice donor and allow for splicing regulation. These results indicate that while the majority of GC-AG introns appear to be constitutively spliced and have no evolutionary constraints to prevent them from being GT-AG introns, a subset of GC-AG introns is involved in alternative splicing and the C at the +2 position of these introns can have an important role in splicing regulation.     

Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans

The spindle-assembly checkpoint ensures that, during mitosis and meiosis, chromosomes do not segregate until they are properly attached to the microtubules of the spindle. Here we show that mdf-1 and mdf-2 are components of the spindle-assembly checkpoint in Caenorhabditis elegans, and are essential for the long-term survival and fertility of this organism. Loss of function of either of these genes leads to the accumulation of a variety of defects, including chromosome abnormalities, X-chromosome non-disjunction or loss, problems in gonad development, and embryonic lethality. Antibodies that recognize the MDF-2 protein localize to nuclei of the cleaving embryo in a cell-cycle-dependent manner. mdf-1, a gene encoding a product that interacts with MDF-2, is required for cell-cycle arrest and proper chromosome segregation in premeiotic germ cells treated with nocodazole, a microtubule-depolymerizing agent. In the absence of mdf gene products, errors in chromosome segregation arise and accumulate, ultimately leading to genetic lethality.     

N-Glycans of Caenorhabditis elegans are specific to developmental stages

We have examined the N-glycans present during the developmental stages of Caenorhabditis elegans using two approaches, 1) a combination of permethylation followed by MALDI-TOF mass spectrometry (MS) and 2) derivatization with 2-aminobenzamide followed by separation by high-performance liquid chromatography and analyses by MALDI-TOF MS, post source decay (PSD) MS, and MALDI-QoTOF MS/MS. The N-glycan profile of each developmental stage (Larva 1, Larva 2, Larva 3, Larva 4, and Dauer and adult) appears to be unique. The pattern of complex N-glycans was stage-specific with the general trend of number and abundance of glycans being Dauer approximately = L1 > adult approximately = L4 > L3 approximately = L2. Dauer larvae contained complex N-glycans with higher molecular masses than those seen in other stages. MALDI-QoTOF MS/MS of Hex4HexNAc4 showed an N-acetyllac-tosamine substitution not previously observed in C. elegans. Phosphorylcholine (Pc)-substituted glycans were also found to be stage-specific. Higher molecular weight Pc-containing glycans, including fucose-containing ones such as difucosyl Pc-glycan (Pc1dHex2Hex5HexNAc6) seen in Dauer larvae, have not been observed in any organism. Pc2Hex4HexNAc3, from Dauer larvae, when subjected to PSD MS analyses, showed Pc may substitute both core and terminally linked GlcNAc; no such structure has previously been reported in any organism. C. elegans-specific fucosyl and native methylated glycans were found in all developmental stages. Taken together, the above results demonstrate that in-depth investigation of the role of the above N-glycans during C. elegans development should lead to a better understanding of their significance and the ways that they may govern interactions, both within the organism during development and between the mobile nematode and its pathogens.