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C. elegansis a free-living nematode that must adapt to a wide range of environments including both aerobic and anaerobic conditions. To survive in low oxygen,C. eleganscan use an unusual form of anaerobic respiration that relies on rhodoquinone (RQ) as an alternative electron carrier. Parasitic nematodes like hookworm and whipworm also require rhodoquinone-dependent metabolism (RQDM) to survive in the highly anaerobic conditions in the human gut. Understanding how RQDM is regulated inC. elegansmay thus identify new ways to combat these closely-related major human pathogens. We previously established a simple movement-based assay for RQDM inC. elegans. In this study, we tested a panel of wild-type isolates ofC. elegansin our RQDM assay and find substantial variation in their ability to use RQDM. We carried out a genome-wide association study (GWAS) to identify loci that affect RQDM — this identified a single major QTL on the right arm of Chromosome III. We used RNAi to test almost all genes within the QTL region for involvement in RQDM and found one gene,wah-1, that strongly modulates RQDM-dependent recovery inC. elegans. WAH-1 is a mitochondrial flavoprotein that affects the electron transport chain, consistent with a role in RQDM. We show thatwah-1expression varies between isolates due to major changes inwah-1transcript structures and this correlates tightly with variation in RQDM. Finally, we show that there is similar complexity towah-1transcription in parasitic nematodes and thatwah-1transcript structures change as parasites shift from aerobic to anaerobic, RQ- requiring metabolism. We thus conclude that reducedwah-1expression correlates with increased ability to survive in conditions where RQDM is essential.

Soil-transmitted helminths (STHs) including Ascaris, hookworm, and whipworm are major human pathogens infecting over a billion people worldwide1,2. There are few existing classes of anthelmintics and resistance is increasing3–5 — there is thus an urgent need for new classes of these drugs. Here we focus on identifying compounds that interfere with the unusual anaerobic metabolism that STHs use to survive the highly hypoxic conditions of the host gut6–9. This requires rhodoquinone (RQ), a quinone electron carrier that is not made or used by the STH hosts10. We previously showed that C. elegans also uses this rhodoquinone-dependent metabolism (RQDM)11 and established a high throughput assay for RQDM11. We screened a collection of 480 natural products for compounds that kill worms specifically when they rely on RQDM — these 480 are representatives of a full library of ~25,000 natural products and derivatives12,13. We identify several classes of compound including a novel family of species selective inhibitors of Complex I. These Complex I inhibitors are based on a benzimidazole core but unlike commercial benzimidazole anthelmintics they do not target microtubules14–17. We screened over 1,200 benzimidazoles and identify the key structural requirements for species selective Complex I inhibition. We suggest that these novel benzimidazole species-selective Complex I inhibitors may be potential anthelmintics.

Soil transmitted helminths (STHs) are major human pathogens that infect over a billion people. Resistance to current anthelmintics is rising and new drugs are needed. Here we combine multiple approaches to find druggable targets that are essential for RQ-dependent metabolism, an unusual form of anaerobic metabolism which STHs need to survive in their host. We identified 25 genes predicted to act in RQ-dependent metabolism from sensing hypoxia to RQ synthesis — this includes components of the kynurenine pathway we previously showed to be essential for RQ synthesis (Del Borrello et al., 2019). We found 9 genes to be required — since all have host orthologues, we used comparative genomics and structural modeling to identify those with helminth-specific active sites and found 4 such targets. These 4 high confidence targets open up the possibility of in silico screens to identify STH-specific inhibitors of these enzymes as new anthelmintics.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize twocoq-2splice forms,coq-2aandcoq-2e, and thecoq-2e-specific exon is only found in species that synthesize RQ. We show that inCaenorhabditis elegansCOQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing ofcoq-2.

Parasitic nematodes infect one quarter of the world's population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery., Screening for new anthelmintic compounds that are active against parasitic nematodes is costly and labour intensive. Here, the authors use the non-parasitic nematode Caenorhabditis elegans to identify 30 anthelmintic lead compounds in an effective and cost-efficient manner.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In normoxia, they use ubiquinone (UQ), but in anaerobic conditions inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. We previously showed the switch from UQ to RQ synthesis is driven by a change of substrates by the polyprenyltransferase COQ-2 (Del Borrello et al., 2019; Roberts Buceta et al., 2019); however, the mechanism of substrate selection is not known. Here, we show helminths synthesize two coq-2 splice forms, coq-2a and coq-2e, and the coq-2e-specific exon is only found in species that synthesize RQ. We show that in Caenorhabditis elegans COQ-2e is required for efficient RQ synthesis and survival in cyanide. Importantly, parasites switch from COQ-2a to COQ-2e as they transit into anaerobic environments. We conclude helminths switch from UQ to RQ synthesis principally via changes in the alternative splicing of coq-2.

Genes encoding essential components of core cellular processes are typically highly conserved across eukaryotes. However, a small proportion of essential genes are highly taxonomically restricted; there appear to be no similar genes outside the genomes of highly related species. What are the functions of these poorly characterized taxonomically restricted genes (TRGs)? Systematic screens in Saccharomyces cerevisiae and Caenorhabditis elegans previously identified yeast or nematode TRGs that are essential for viability and we find that these genes share many molecular features, despite having no significant sequence similarity. Specifically, we find that those TRGs with essential phenotypes have an expression profile more similar to highly conserved genes, they have more protein–protein interactions and more protein disorder. Surprisingly, many TRGs play central roles in chromosome segregation; a core eukaryotic process. We thus find that genes that appear to be highly evolutionarily restricted do not necessarily play roles in species-specific biological functions but frequently play essential roles in core eukaryotic processes.

Parasitic helminths use two benzoquinones as electron carriers in the electron transport chain. In aerobic environments they use ubiquinone (UQ) but in anaerobic environments inside the host, they require rhodoquinone (RQ) and greatly increase RQ levels. The switch to RQ synthesis is driven by substrate selection by the polyprenyltransferase COQ-2 but the mechanisms underlying this substrate choice are unknown. We found that helminths make twocoq-2isoforms,coq-2aandcoq-2e, by alternative splicing. COQ-2a is homologous to COQ2 from other eukaryotes but the COQ-2e-specific exon is only found in species that make RQ and its inclusion changes the enzyme core. We show COQ-2e is required for RQ synthesis and for survival in cyanide inC. elegans. Crucially, we see a switch from COQ-2a to COQ-2e as parasites transition into anaerobic environments. We conclude that under anaerobic conditions helminths switch from UQ to RQ synthesis via alternative splicing ofcoq-2.

Parasitic helminths infect over a billion humans. To survive in the low oxygen environment of their hosts, these parasites use unusual anaerobic metabolism — this requires rhodoquinone (RQ), an electron carrier that is made by very few animal species. Crucially RQ is not made or used by any parasitic hosts and RQ synthesis is thus an ideal target for anthelmintics. However, little is known about how RQ is made and no drugs are known to block RQ synthesis. C. elegans makes RQ and can use RQ-dependent metabolic pathways — here, we use C. elegans genetics to show that tryptophan degradation via the kynurenine pathway is required to generate the key amine-containing precursors for RQ synthesis. We show that C. elegans requires RQ for survival in hypoxic conditions and, finally, we establish a high throughput assay for drugs that block RQ-dependent metabolism. This may drive the development of a new class of anthelmintic drugs. This study is a key first step in understanding how RQ is made in parasitic helminths., eLife digest Parasitic worms infect more than a billion people worldwide, using a range of tricks to survive inside the human body. Some species can live for weeks inside the gut, a place with almost no oxygen. Yet exactly how they manage this is remains unclear. Scientists know that parasitic worms have an unusual way of making chemical energy when oxygen levels drop. Like human cells, worm cells use a series of molecular complexes called the electron transport chain. As electrons pass along the chain, they drive the production of chemical energy. Normally, oxygen sits at the end of the chain to receive the electrons. But, when there is no oxygen, almost all animals stop using the electron transport chain. A few animals can continue to use it by using other molecules to receive the final electrons instead of oxygen. To do that, they need a special electron carrier and, in worms, this electron carrier is rhodoquinone. Human cells do not use rhodoquinone, making it a prime target for drug design. If a drug could block rhodoquinone production, it might be able to stop worms surviving in the human intestines without harming the patient’s own cells. Yet, even though the scientific community has known about rhodoquinone for more than 50 years, it remains unclear how worms make this molecule. To find out, Del Borrello et al. examined the laboratory worm Caenorhabditis elegans. This worm is not a parasite, but it does make rhodoquinone. Del Borrello et al. developed a new way to study rhodoquinone production by blocking the normal route of the electron transport chain with cyanide. This causes the worms to switch to using rhodoquinone and is cheaper than raising the worms in low oxygen, making it easier to conduct high-throughput screening. A combination of chemistry and information from other species made it possible to identify candidate genes responsible for the production of rhodoquinone. Worms with faults in these genes revealed the key building blocks of rhodoquinone, and the early steps in its production. Removing any one of the genes made it harder for the worms to survive without oxygen. Although there are already effective drugs that kill parasitic worms, resistance is growing. A better understanding of rhodoquinone could lead to a new class of drugs to help control this major problem in global health. A drug that blocks any one of the production steps of rhodoquinone might be a future candidate for a new anti-parasitic worm therapy.

Parasitic helminths infect over a billion humans. To survive in the low oxygen environment of their hosts, these parasites use unusual anaerobic metabolism. This requires Rhodoquinone (RQ), an electron carrier that is made by very few animal species — crucially it is not present in any parasitic hosts. RQ synthesis is thus an ideal target for anthelmintics but little is known about how RQ is made and no drugs are known to block RQ synthesis.C.elegansmakes RQ and can use RQ-dependent metabolic pathways — here, we useC.elegansgenetics to identify the pathway for RQ synthesis and show thatC.elegansrequires RQ for survival in hypoxic conditions. Finally, we establish a robust assay for drugs that block RQ-dependent metabolism. This study identifies for the first time how RQ is made in any animal and establishes a novel assay that can drive the development of a new class of anthelmintic drugs.

Apoptotic death of cells damaged by genotoxic stress requires regulatory input from surrounding tissues. The C. elegans scaffold protein KRI-1, ortholog of mammalian KRIT1/CCM1, permits DNA damage-induced apoptosis of cells in the germline by an unknown cell non-autonomous mechanism. We reveal that KRI-1 exists in a complex with CCM-2 in the intestine to negatively regulate the ERK-5/MAPK pathway. This allows the KLF-3 transcription factor to facilitate expression of the SLC39 zinc transporter gene zipt-2.3, which functions to sequester zinc in the intestine. Ablation of KRI-1 results in reduced zinc sequestration in the intestine, inhibition of IR-induced MPK-1/ERK1 activation, and apoptosis in the germline. Zinc localization is also perturbed in the vasculature of krit1−/− zebrafish, and SLC39 zinc transporters are mis-expressed in Cerebral Cavernous Malformations (CCM) patient tissues. This study provides new insights into the regulation of apoptosis by cross-tissue communication, and suggests a link between zinc localization and CCM disease., Cerebral Cavernous Malformations (CCM) are often caused by mutations in CCM1/KRIT1. Here, Chapman et al. elegantly show that the CCM complex promotes apoptosis by regulating zinc homeostasis and storage via a conserved mechanism that likely generates the pathological defects observed in CCM.

Many drugs act very rapidly — they can turn on or off their targets within minutes in a whole animal. What are the acute effects of drug treatment and how does an animal respond to these? We developed a simple assay to measure the acute effects of drugs on C. elegans movement and examined the effects of a range of compounds including neuroactive drugs, toxins, environmental stresses and novel compounds on worm movement over a time period of 3 hr. We found a wide variety of acute responses. Many compounds cause rapid paralysis which may be permanent or followed by one or more recovery phases. The recoveries are not the result of some generic stress response but are specific to the drug e.g., recovery from paralysis due to a neuroactive drug requires neurotransmitter pathways whereas recovery from a metabolic inhibitor requires metabolic changes. Finally, we also find that acute responses can vary greatly across development and that there is extensive natural variation in acute responses. In summary, acute responses are sensitive probes of the ability of biological networks to respond to drug treatment and these responses can reveal the action of unexplored pathways.

Many drugs act very rapidly — they can turn on or off their targets within minutes in a whole animal. What are the acute effects of drug treatment and how does an animal respond to these? We developed a simple assay to measure the acute effects of drugs onC.elegansmovement and examined the effects of a range of compounds including neuroactive drugs, toxins, environmental stresses and novel compounds on worm movement over a time period of 3 hours. We find that many treatments show complex acute responses — a phase of rapid paralysis is followed by one or more recovery phases. The recoveries are not the result of some generic stress response but are specific to the drug e.g. recovery from paralysis due to a neuroactive drug requires neurotransmitter pathways whereas recovery from a metabolic inhibitor requires metabolic changes. Finally, we also find that acute responses can vary greatly across development and that there is extensive and complex natural variation in acute responses. In summary, acute responses are sensitive probes of the ability of biological networks to respond to drug treatment and these responses can reveal the action of unexplored pathways.Author SummaryDrugs are powerful tools that let us switch on or off key pathways in whole animals and watch the effects. Here we set up a simple assay to measure how drugs affect the movement of the simple nematodeC.elegans— crucially, we look how those responses change over time. We find that worms have complex responses to many different drugs — they go through clear phases of paralysis and recovery. The recovery from the initial effect of any drug is not due to a generic stress response but is specific to the way each drug acts. We find for example that worms can recover from paralysis driven by one neurotransmitter pathway by activating a different neurotransmitter pathway or from paralysis caused by loss of one metabolic pathway by turning on a second one. These complex responses show how the basic genetic networks that are needed for normal behaviour and function can rewire and respond to the effect of many drugs. Importantly, the responses can vary in many ways — different aged worms or different individuals can have different responses and capturing how drug responses change over time is critical to dissect this complexity.

Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation-mass spectrometry, CF-MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF-MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container (https://hub.docker.com/r/baderlab/bio-epic/).

Background In addition to DNA, gametes contribute epigenetic information in the form of histones and non-coding RNA. Epigenetic programs often respond to stressful environmental conditions and provide a heritable history of ancestral stress that allows for adaptation and propagation of the species. In the nematode C. elegans, defective epigenetic transmission often manifests as progressive germline mortality. We previously isolated sup-46 in a screen for suppressors of the hexosamine pathway gene mutant, gna-2(qa705). In this study, we examine the role of SUP-46 in stress resistance and progressive germline mortality. Results We identified SUP-46 as an HNRNPM family RNA-binding protein, and uncovered a highly novel role for SUP-46 in preventing paternally-mediated progressive germline mortality following mating. Proximity biotinylation profiling of human homologs (HNRNPM, MYEF2) identified proteins of ribonucleoprotein complexes previously shown to contain non-coding RNA. Like HNRNPM and MYEF2, SUP-46 was associated with multiple RNA granules, including stress granules, and also formed granules on active chromatin. SUP-46 depletion disrupted germ RNA granules and caused ectopic sperm, increased sperm transcripts, and chronic heat stress sensitivity. SUP-46 was also required for resistance to acute heat stress, and a conserved “MYEF2” motif was identified that was needed for stress resistance. Conclusions In mammals, non-coding RNA from the sperm of stressed males has been shown to recapitulate paternal stress phenotypes in the offspring. Our results suggest that HNRNPM family proteins enable stress resistance and paternally-mediated epigenetic transmission that may be conserved across species. Electronic supplementary material The online version of this article (doi:10.1186/s12915-017-0398-y) contains supplementary material, which is available to authorized users.

Parasitic nematodes negatively impact human and animal health worldwide. The market withdrawal of nematicidal agents due to unfavourable toxicities has limited the available treatment options. In principle, co-administering nematicides at lower doses along with molecules that potentiate their activity could mitigate adverse toxicities without compromising efficacy. Here, we screened for new small molecules that interact with aldicarb, which is a highly effective treatment for plant-parasitic nematodes whose toxicity hampers its utility. From our collection of 638 worm-bioactive compounds, we identified 20 molecules that interact positively with aldicarb to either kill or arrest the growth of the model nematode Caenorhabditis elegans. We investigated the mechanism of interaction between aldicarb and one of these novel nematicides called wact-86. We found that the carboxylesterase enzyme GES-1 hydrolyzes wact-86, and that the interaction is manifested by aldicarb’s inhibition of wact-86’s metabolism by GES-1. This work demonstrates the utility of C. elegans as a platform to search for new molecules that can positively interact with industrial nematicides, and provides proof-of-concept for prospective discovery efforts., Author summary Many nematicides that have been used to kill plant and animal parasitic nematodes are being phased out over concerns of toxicity to humans. One potential solution to reduce toxicity is to use the nematicide at a lower concentration in combination with a second compound that together will produce a synergistic killing effect. That is, the use of either molecule alone at low concentrations is non-lethal, but when used together at these same concentrations, the cocktail is lethal. This strategy has two benefits. First, the killing effect is concentrated at the site of use and as the two molecules diffuse from the targeted site, toxicity is negated. Second, less of the toxic molecule is needed and therefore less is dispersed into the environment. Here, we describe our use of a model nematode called C. elegans to search for molecules that interact with aldicarb, which is one of the nematicides being phased out by environmental agencies. We identified 20 compounds that interact with aldicarb and describe how one of these, called wact-86, functions with aldicarb to kill worms. Our work provides proof-of-principle that C. elegans is a useful model for identifying compounds that positively interact with industrial nematicides and for understanding the nature of such interactions.

Alternative splicing is important for the development and function of the nervous system, but little is known about the differences in alternative splicing between distinct types of neurons. Furthermore, the factors that control cell-type-specific splicing and the physiological roles of these alternative isoforms are unclear. By monitoring alternative splicing at single-cell resolution in Caenorhabditis elegans, we demonstrate that splicing patterns in different neurons are often distinct and highly regulated. We identify two conserved RNA-binding proteins, UNC-75/CELF and EXC-7/Hu/ELAV, which regulate overlapping networks of splicing events in GABAergic and cholinergic neurons. We use the UNC-75 exon network to discover regulators of synaptic transmission and to identify unique roles for isoforms of UNC-64/Syntaxin, a protein required for synaptic vesicle fusion. Our results indicate that combinatorial regulation of alternative splicing in distinct neurons provides a mechanism to specialize metazoan nervous systems.

Alternative splicing (AS) is the process by which multiple mRNA transcripts are produced from a single precursor transcript through the differential utilization of splice sites. Alternative splicing is one of the key mechanisms that have evolved in metazoans to generate increased transcriptome complexity and recent studies estimate that greater than 95% of human multi-exon genes express multiple splice isoforms (Pan et al. 2008; Wang et al. 2008). Moreover, alternatively spliced exons are often differentially regulated across tissues and during development, suggesting that individual isoforms may serve specific spatial or temporal roles (Hartmann and Valcarcel 2009; Licatalosi and Darnell 2010; Nilsen and Graveley 2010). The importance of proper regulation of AS during development has been demonstrated in many different instances; one particularly well-studied example is that of the sex determination pathway in Drosophila. In this pathway, the female-specific expression of a splicing regulator transformer stimulates the inclusion of exons in transcripts of the doublesex and fruitless transcription factor genes (Lopez 1998; Forch and Valcarcel 2003). The female-specific isoforms of these transcription factors subsequently activate the expression of genes required for female development, while the male-specific variants induce a gene expression program important for male differentiation (Dulac 2005; Shirangi and McKeown 2007). Similar spatio-temporally regulated AS networks are likely to exist in metazoans. The characterization of these AS networks, and their integration with other layers of gene regulation, will be necessary for a more complete understanding of development (Blencowe 2006). The nematode C. elegans, with its amenability for genetic manipulations, defined cell lineage, simple but extensively differentiated nervous system, conserved splicing machinery, and established molecular and cell biological resources, provides an attractive yet relatively under-explored model organism with which to study the regulation and functions of AS in vivo. Tissue or developmental stage-specific AS events have been identified in approximately 70 C. elegans genes which, like those identified in vertebrates, are controlled by combinatorial interactions between splicing factors (Kuroyanagi et al. 2007; Barberan-Soler and Zahler 2008; Ohno et al. 2008). These studies, while informative, were limited to the analysis of isoforms represented in expressed sequence tag (EST)/cDNA libraries, which currently do not provide a full coverage of the transcriptome. The advent of deep sequencing technologies has now enabled transcriptome analyses at unprecedented levels of sensitivity and precision, and these have recently been applied in C. elegans (Hillier et al. 2009; Ramani et al. 2009; Mangone et al. 2010). However, due in part to limited depth of coverage of existing data sets and the ongoing development of analytical software, it has remained a major challenge to systematically categorize and quantify the relative usage of isoforms across development for both known and novel AS events. In this study, we combined high-throughput sequencing of mRNA (RNA-seq) and quantitative AS microarray profiling to provide a high resolution survey of splicing across C. elegans development. We have identified thousands of new AS events and hundreds of isoforms that are differentially expressed during development. Additionally, we identified candidate cis-elements that likely play a role in regulating these events. To facilitate the use of these data, we have created the “C. elegans Splice Browser,” a web resource in which all of the data in the present study are made accessible. This resource, which represents the most complete set of splice variants reported to date, should serve as a valuable basis for focused analyses of the functional importance of individual splice isoforms in vivo.

The rate of RNA polymerase II (Pol II) elongation can influence splice site selection in nascent transcripts, yet the extent and physiological relevance of this kinetic coupling between transcription and alternative splicing (AS) is not well understood. We performed experiments to perturb Pol II elongation and then globally compared AS patterns with genome-wide Pol II occupancy. RNA binding and RNA processing functions were significantly enriched among the genes with Pol II elongation inhibition-dependent changes in AS. Under conditions that interfere with Pol II elongation, including cell stress, increased Pol II occupancy was detected in the intronic regions flanking the alternative exons in these genes, and these exons generally became more included. A disproportionately high fraction of these exons introduced premature termination codons that elicited nonsense-mediated mRNA decay (NMD), thereby further reducing transcript levels. Our results provide evidence that kinetic coupling between transcription, AS, and NMD affords a rapid mechanism by which cells can respond to changes in growth conditions, including cell stress, to coordinate the levels of RNA processing factors with mRNA levels.

G protein-coupled receptors (GPCRs) have a key role in many biological processes and are important drug targets for many human diseases. Therefore, understanding the molecular interactions between GPCRs and their ligands would improve drug design. Here, we describe an approach that allows the rapid identification of functional agonists expressed in bacteria. TransgenicCaenorhabditis elegansexpressing the human chemokine receptor 5 (CCR5) in nociceptive neurons show avoidance behavior on encounter with the ligand MIP-1α and avoid feeding onEscherichia coliexpressing MIP-1α compared with control bacteria. This system allows a simple activity screen, based on the distribution of transgenic worms in a binary food-choice assay, without a requirement for protein purification or tagging. By using this approach, a library of 68 MIP-1α variants was screened, and 13 critical agonist residues involved in CCR5 activation were identified, four of which (T8, A9, N22, and A25) have not been described previously, to our knowledge. Identified residues were subsequently validated in receptor binding assays and by calcium flux assays in mammalian cells. This approach serves not only for structure/function studies as demonstrated, but may be used to facilitate the discovery of agonists within bacterial libraries.

Parasitic nematodes infect one quarter of the world’s population and impact all humans through widespread infection of crops and livestock. Resistance to current anthelmintics has prompted the search for new drugs. Traditional screens that rely on parasitic worms are costly and labour intensive and target-based approaches have failed to yield novel anthelmintics. Here, we present our screen of 67,012 compounds to identify those that kill the non-parasitic nematode Caenorhabditis elegans. We then rescreen our hits in two parasitic nematode species and two vertebrate models (HEK293 cells and zebrafish), and identify 30 structurally distinct anthelmintic lead molecules. Genetic screens of 19 million C. elegans mutants reveal those nematicides for which the generation of resistance is and is not likely. We identify the target of one lead with nematode specificity and nanomolar potency as complex II of the electron transport chain. This work establishes C. elegans as an effective and cost-efficient model system for anthelmintic discovery.

We describe a protocol for performing RNA interference (RNAi) screens in Caenorhabditis elegans in liquid culture in 96-well plates. The procedure allows a single researcher to set-up and score RNAi experiments at approximately 2,000 genes per day. By comparing RNAi phenotypes between wild-type worms and worms carrying a defined genetic mutation, we have used this protocol to identify synthetic lethal interactions between genes systematically. We also describe how the protocol can be adapted to target two genes simultaneously by combinatorial RNAi.

In multicellular organisms some genes are expressed in essentially all tissues, whereas others are expressed predominantly in only one or a few tissues. In this study, we investigate the relationship between the tissue-specificity of gene expression and the type of protein encoded. We find that many protein domains are found to be enriched in either tissue-specific or widely expressed genes. Domains enriched in tissue-specific genes tend to be metazoan-specific; these same domains are also enriched in genes that are not essential for cell viability. These findings identify families of proteins that are probably used in the development or terminal differentiation of many different tissue types.

Cells are controlled by the complex and dynamic actions of thousands of genes. With the sequencing of many genomes, the key problem has shifted from identifying genes to knowing what the genes do; we need a framework for expressing that knowledge. Even the most rigorous attempts to construct ontological frameworks describing gene function (e.g., the Gene Ontology project) ultimately rely on manual curation and are thus labor-intensive and subjective. But an alternative exists: the field of functional genomics is piecing together networks of gene interactions, and although these data are currently incomplete and error-prone, they provide a glimpse of a new, probabilistic view of gene function. We outline such a framework, which revolves around a statistical description of gene interactions derived from large, systematically compiled data sets. In this probabilistic view, pleiotropy is implicit, all data have errors and the definition of gene function is an iterative process that ultimately converges on the correct functions. The relationships between the genes are defined by the data, not by hand. Even this comprehensive view fails to capture key aspects of gene function, not least their dynamics in time and space, showing that there are limitations to the model that must ultimately be addressed.

Genetic screens are powerful tools for identifying the genes involved in specific biological processes. At last, RNA interference makes large-scale screens possible in mammalian cells.

To initiate studies on how protein-protein interaction (or “interactome”) networks relate to multicellular functions, we have mapped a large fraction of the Caenorhabditis elegans interactome network. Starting with a subset of metazoan-specific proteins, more than 4000 interactions were identified from high-throughput, yeast two-hybrid (HT=Y2H) screens. Independent coaffinity purification assays experimentally validated the overall quality of this Y2H data set. Together with already described Y2H interactions and interologs predicted in silico , the current version of the Worm Interactome (WI5) map contains ∼5500 interactions. Topological and biological features of this interactome network, as well as its integration with phenome and transcriptome data sets, lead to numerous biological hypotheses.

RNA interference (RNAi) has been rapidly adopted as a general method for inhibiting gene expression in most laboratory organisms. This paper discusses how libraries of RNAi reagents are being used to perform genome-wide reverse genetic screens in both model organisms and mammalian cells. Guidelines for designing effective small interfering RNAs and appropriate controls for mammalian RNAi experiments will also be discussed.

Regulation of body fat storage involves signalling between centres that regulate feeding in the brain and sites of fat storage and use in the body1,2. Here we describe an assay for analysing fat storage and mobilization in living Caenorhabditis elegans. By using RNA-mediated interference (RNAi)3,4 to disrupt the expression of each of the 16,757 worm genes, we have systematically screened the C. elegans genome for genes necessary for normal fat storage. We identify 305 gene inactivations that cause reduced body fat and 112 gene inactivations that cause increased fat storage. Analysis of the fat-reducing gene inactivations in insulin, serotonin and tubby signalling mutants of C. elegans, which have increased body fat, identifies a core set of fat regulatory genes as well as pathway-specific fat regulators. Many of the newly identified worm fat regulatory genes have mammalian homologues, some of which are known to function in fat regulation. Other C. elegans fat regulatory genes that are conserved across animal phylogeny, but have not previously been implicated in fat storage, may point to ancient and universal features of fat storage regulation, and identify targets for treating obesity and its associated diseases.

To explore the role of mitochondrial activity in the aging process, we have lowered the activity of the electron transport chain and adenosine 5′-triphosphate (ATP) synthase with RNA interference (RNAi) in Caenorhabditis elegans. These perturbations reduced body size and behavioral rates and extended adult life-span. Restoring messenger RNA to near-normal levels during adulthood did not elevate ATP levels and did not correct any of these phenotypes. Conversely, inhibiting respiratory-chain components during adulthood only did not reset behavioral rates and did not affect life-span. Thus, the developing animal appears to contain a regulatory system that monitors mitochondrial activity early in life and, in response, establishes rates of respiration, behavior, and aging that persist during adulthood.

The genetic analysis of life span has revealed many interesting genes and pathways; however, our understanding of aging has been limited by the lack of a way to assay the aging process itself. Here we show that the tissues of aging worms have a characteristic appearance that is easy to recognize and quantify using Nomarski optics. We have used this assay to determine whether life-span mutations affect the rate of aging, to identify animals that age more rapidly than normal, and to infer the cause of death in C. elegans. Mutations that reduce insulin/IGF-1 signaling double the life span of C. elegans, and we find that tissue decline is slowed in these mutants. Thus this endocrine system appears to influence the rate at which tissues age. This effect extends even to the germline, which is the only mitotically active tissue in the adult. We find that Nomarski microscopy also allows a ready distinction between short-lived mutants that age more rapidly than normal and those that are simply sick, and we have identified an RNAi clone that confers a dramatic rapid-aging phenotype. This clone encodes the C. elegans heat-shock factor (HSF), a transcription factor that regulates the response to heat and oxidative stress. This suggests that heat-shock proteins, many of which act as chaperones, may function in normal animals to slow the rate of aging. Finally, we have identified a cause of death of C. elegans: namely, proliferating bacteria. This suggests that increased susceptibility to bacterial infections contributes to mortality in these animals, just as it does in humans.

Genetic screens in the hermaphrodite nematode worm Caenorhabditis elegans resulted in the identification of the basal conserved machinery of apoptosis, arguably the single most important finding for our understanding of cell death. The last two years have seen enormous progress in the elucidation of the molecular interactions that lie at the heart of this conserved machinery, along with major insights both into how cell death is activated in the worm and into the mechanism of recognition and engulfment of the cell corpses. In this review, I set out the current models of cell death regulation and execution in C. elegans, focussing in particular on the similarities between cell death in C. elegans and vertebrates. Finally, I attempt to highlight key areas for future progress in cell death research in C. elegans and explore additional ways in which the worm can be used to understand the regulation of cell death in mammals.

Three studies in Science and Cell identify ∼2,000 essential genes in human cell lines using CRISPR-Cas9 and retroviral gene-trap genetic screens.

Although two related species may have extremely similar phenotypes, the genetic networks underpinning this conserved biology may have diverged substantially since they last shared a common ancestor. This is termed Developmental System Drift (DSD) and reflects the plasticity of genetic networks. One consequence of DSD is that some orthologous genes will have evolved different in vivo functions in two such phenotypically similar, related species and will therefore have different loss of function phenotypes. Here we report an RNAi screen in C. elegans and C. briggsae to identify such cases. We screened 1333 genes in both species and identified 91 orthologues that have different RNAi phenotypes. Intriguingly, we find that recently evolved genes of unknown function have the fastest evolving in vivo functions and, in several cases, we identify the molecular events driving these changes. We thus find that DSD has a major impact on the evolution of gene function and we anticipate that the C. briggsae RNAi library reported here will drive future studies on comparative functional genomics screens in these nematodes., Author Summary Although two related species may appear similar, the genetic pathways that underpin this shared biology may have drifted and changed. This phenomenon is known as Developmental System Drift (DSD). One consequence of DSD is that equivalent genes may play different roles in phenotypically similar, related species but there have been no systematic studies to examine this. How often do genes have different functions in similar species? Are certain genes more likely to change functions? Finally, what are the molecular changes that drive this? Here, we compare the effects of reducing the levels of over 1300 different genes in two species of nematode worm. These worms are very similar— they live in the same ecological niche, and have near-identical development and behavior. We find that over 25% of conserved genes have different functions in these two species, showing that DSD has a major impact on how gene function evolves. Intriguingly, we find that genes that have arisen recently are most likely to change functions and that this is often driven by changes in their expression. This is the first systematic comparison of loss of function phenotypes in related species and sheds light on how genetic pathways rewire during DSD.

Cysteine proteases of the ICE/CED‐3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila . We report here the identification and characterization of drICE, a D.melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N‐terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr . drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.

Background: Three principal genes are involved in developmental programmed cell death (PCD) in the nematode worm Caenorhabditis elegans . The ced-3 and ced-4 genes are both required for each PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes in cells that are destined to survive. Vertebrate homologues of both ced-3 and ced-9 have been identified as the genes encoding the caspase cysteine proteases and the Bcl-2 family, respectively. In contrast, no vertebrate homologue of ced-4 is known. The CED-3/caspases are important effectors of apoptosis that are presumed to act by cleaving specific target substrates. However, the molecular functions of the CED-9/Bcl-2 and CED-4 proteins are unknown. The unicellular yeast Schizosaccharomyces pombe shares many general cellular properties with metazoa, but has no identified cell suicide machinery. We have therefore used S. pombe as a naive model cell system in which to examine the biological effects of cell-death proteins. Results: Induction of wild-type ced-4 expression in S. pombe resulted in rapid focal chromatin condensation and lethality. Mutation of the putative nucleotide-binding P-loop motif of CED-4 (K165Q) eliminated the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggested that the phenotype may result from an intrinsic activity of CED-4. Co-expression of ced-9 prevented CED-4-induced chromatin condensation and lethality, and caused the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. Conclusions: Using S. pombe as a model system in which to assay CED-4 function, we have identified a potential direct role for CED-4 in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. The CED-9-mediated rescue of CED-4-induced lethality in this system and the interaction of the two proteins in the yeast two-hybrid analysis suggest that CED-9 inhibits CED-4 action by direct physical association.

RNA-binding proteins are key regulators of gene expression, yet only a small fraction have been functionally characterized. Here we report a systematic analysis of the RNA motifs recognized by RNA-binding proteins, encompassing 205 distinct genes from 24 diverse eukaryotes. The sequence specificities of RNA-binding proteins display deep evolutionary conservation, and the recognition preferences for a large fraction of metazoan RNA-binding proteins can thus be inferred from their RNA-binding domain sequence. The motifs that we identify in vitro correlate well with in vivo RNA-binding data. Moreover, we can associate them with distinct functional roles in diverse types of post-transcriptional regulation, enabling new insights into the functions of RNA-binding proteins both in normal physiology and in human disease. These data provide an unprecedented overview of RNA-binding proteins and their targets, and constitute an invaluable resource for determining post-transcriptional regulatory mechanisms in eukaryotes.

Apoptosis is a common feature of the nervous system, occurring physiologically during development and pathologically in several diseases. In view of the latter, pathways that regulate apoptosis in neurones are of particular interest, and recent advances in this field implicate several signalling pathways in the induction of apoptosis, after withdrawal of nerve growth factor. Recent information drawn from both mammalian and invertebrate models enables us to assign components of the apoptotic pathway as either regulators or effectors.

Systems biology has been central to Caenorhabditis elegans research from the first steps of establishing the worm as a major model system through to the post-genomics era. Concepts such as completeness have been central to the philosophy of the worm community from its inception, and the standard systems approach to analyzing biology has been hard-wired into C. elegans research: first find all the components, then map all their connections, and finally examine how the network-level properties of the system explain the biological behavior of the organism. In this chapter, we first outline the initial phase of worm research from the seminal achievements in the worm in the pre-genomics era to the completion of the full genome sequence in 1998. At that point, both the anatomical phenotype and the genotype of the worm were completely known, and the central part of this chapter will discuss the various systems level approaches to understand how the instructions encoded in the 97Mb genome are interpreted to generate the 959 cells of the adult worm. This includes separate sections on genetic screens using both mutagens and RNA-mediated interference, RNA expression and gene regulation, phenotypic variation among isogenic individuals, proteomics approaches, and a section illustrating how computational approaches can integrate diverse large-scale datasets to uncover the molecular machineries that underpin worm biology. Finally, we highlight approaches that have received less attention from the C. elegans community, but that hold some promise as part of the quest to achieve a ‘complete’ understanding this tractable organism. These include dynamic and mechanistic modeling, the interplay between mechanical and biochemical processes, and the linking of natural genetic and phenotypic variation.

This year's international C. elegans meeting celebrated a Nobel prize with a multitude of new research developments.