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Accumulation of circular RNAs (circRNAs) during aging occurs on a genome-wide level for multiple organisms, but its significance is unknown. Generating circRNA loss-of-function mutants is difficult because the vast majority of these RNAs are comprised of exons shared with protein-coding mRNAs. In Caenorhabditis elegans, most circRNAs were previously found to accumulate during aging. Two of the most abundant, age-accumulating circRNAs are generated from exon 4 of the crh-1 gene (circ-crh-1). Here, we found that the biogenesis of circ-crh-1 was regulated by the double-stranded RNA-binding protein ADR-1. We identified Reverse Complementary Match (RCM) sequences in introns flanking circ-crh-1. Using CRISPR-Cas9, we deleted the downstream RCM and found that this completely eliminated expression of the circRNA without affecting linear mRNA expression from the crh-1 gene. Remarkably, worms lacking circ-crh-1 exhibited a significantly longer mean lifespan. Lifespan was partially restored to wild type by expression of circ-crh-1 in neural tissues. Widespread transcriptome alterations in circ-crh-1 mutants were identified using RNA-Seq. Moving forward, intronic RCM deletion using CRISPR should be a widely applicable method to identify lifespan-regulating circRNAs in C. elegans.

Dynamic changes in chemoreceptor gene expression levels in sensory neurons is one strategy that an animal can use to modify their responses to dietary changes. However, the mechanisms underlying diet-dependent modulation of chemosensory gene expression are unclear. Here, we show that the expression of the srh-234 chemoreceptor gene localized in a single ADL sensory neuron type of C. elegans is downregulated when animals are fed a Comamonas bacterial diet, but not on an E. coli diet. Remarkably, this diet-modulated effect on srh-234 gene expression levels is dependent on the micronutrient vitamin B12 endogenously produced by Comamonas bacteria. Excess propionate and genetic perturbations in the canonical and shunt propionate breakdown pathways are able to override the repressing effects of vitamin B12 on srh-234 expression. The vitamin B12-mediated regulation of srh-234 expression levels in ADL requires the MEF-2 transcription factor, providing a potential mechanism by which dietary vitamin B12 may transcriptionally tune individual chemoreceptor genes in a single sensory neuron type, which in turn may change animal responses to biologically relevant chemicals in their diet.

kin-29 encodes the C. elegans homolog of mammalian Salt-Inducible Kinases (SIKs). kin-29 mutants are small, have increased propensity to develop into non-reproductive dauer larvae, have reduced chemoreceptor gene expression (Lanjuin and Sengupta, 2002; van der Linden et al.., 2007), have reduced cellular ATP despite increased fat stores, and show reduced sleep (Grubbs et al., 2020). KIN-29 phosphorylates and inhibits the class II histone deacetylase 4 homolog HDA-4 to regulate gene expression in sensory neurons (van der Linden et al., 2007). The longevity phenotype of kin-29 mutants is suppressed by mutations in daf-16 (Lanjuin and Sengupta, 2002), which encodes a forkhead box protein O (FOXO) transcription factor (Kenyon et al., 1993). These results indicated that the increased lifespan of kin-29 mutants may be due to reduced insulin signaling. To determine whether HDA-4 is required for the KIN-29 regulation of lifespan, we performed a survival analysis. As previously reported (Lanjuin and Sengupta, 2002), kin-29(oy38) mutants are long-lived. In contrast, hda-4(oy57) mutants are short lived (Fig. 1). The kin-29hda-4 double mutant longevity phenotype was similar to the phenotype of hda-4 single mutants (Fig. 1), suggesting that hda-4 acts downstream of kin-29 to regulate lifespan. Together with our previous findings (van der Linden et al., 2007), these genetic results suggest that KIN-29 may regulate lifespan via the action of HDA-4. Since SIKs can inhibit FOXO activity via HDAC4 to regulate gene expression (Mihaylova et al., 2011; Wang et al., 2011), it would be interesting in future studies to test the model that KIN-29/HDA-4 signaling converges on DAF-16/FOXO to modulate gene expression associated with longevity.

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. DuringC. eleganssleep, energetic stores are allocated to non-neural tasks with a resultant drop in the overall fat stores and energy charge. Mutants lacking KIN-29, theC. eleganshomolog of a mammalian Salt-Inducible Kinase (SIK) that signals sleep pressure, have low ATP levels despite high fat stores, indicating a defective response to cellular energy deficits. Liberating energy stores corrects adiposity and sleep defects ofkin-29mutants.kin-29sleep and energy homeostasis roles map to a small number of sensory neurons that act upstream of fat regulation as well as of central sleep-controlling neurons, suggesting hierarchical somatic/neural interactions regulating sleep and energy homeostasis. Genetic interaction betweenkin-29and the histone deacetylasehda-4coupled with subcellular localization studies indicate that KIN-29 acts in the nucleus to regulate sleep. We propose that KIN-29/SIK acts in nuclei of sensory neuroendocrine cells to transduce low cellular energy charge into the mobilization of energy stores, which in turn promotes sleep.HighlightsSleep is associated with fat mobilization and low ATP levelsMetabolic regulation of sleep requires the salt-induced kinase (SIK) homolog KIN-29KIN-29 acts in sensory neurons upstream of sleep-promoting neuronsNuclear localization of KIN-29 is required for the metabolic regulation of sleepA type 2 histone deacetylase acts down stream of KIN-29 in the regulation of sleep.Liberation of energy from fat-storage cells promotes sleep.Beta-oxidation promotes sleep.

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. During Caenorhabditis elegans sleep, energetic stores are allocated to nonneural tasks with a resultant drop in the overall fat stores and energy charge. Mutants lacking KIN-29, the C. elegans homolog of a mammalian Salt-Inducible Kinase (SIK) that signals sleep pressure, have low ATP levels despite high-fat stores, indicating a defective response to cellular energy deficits. Liberating energy stores corrects adiposity and sleep defects of kin-29 mutants. kin-29 sleep and energy homeostasis roles map to a set of sensory neurons that act upstream of fat regulation as well as of central sleep-controlling neurons, suggesting hierarchical somatic/neural interactions regulating sleep and energy homeostasis. Genetic interaction between kin-29 and the histone deacetylase hda-4 coupled with subcellular localization studies indicate that KIN-29 acts in the nucleus to regulate sleep. We propose that KIN-29/SIK acts in nuclei of sensory neuroendocrine cells to transduce low cellular energy charge into the mobilization of energy stores, which in turn promotes sleep., Sleep is intricately connected with metabolism. This study shows that KIN-29, the orthologue of the mammalian salt-inducible kinase (SIK) in the nematode Caenorhabditis elegans, is a key regulator involved in connecting sleep and energy homeostasis.

Background Circular RNAs (CircRNAs) are a newly appreciated class of RNAs that lack free 5′ and 3′ ends, are expressed by the thousands in diverse forms of life, and are mostly of enigmatic function. Ostensibly due to their resistance to exonucleases, circRNAs are known to be exceptionally stable. Previous work in Drosophila and mice have shown that circRNAs increase during aging in neural tissues. Results Here, we examined the global profile of circRNAs in C. elegans during aging by performing ribo-depleted total RNA-seq from the fourth larval stage (L4) through 10-day old adults. Using stringent bioinformatic criteria and experimental validation, we annotated a high-confidence set of 1166 circRNAs, including 575 newly discovered circRNAs. These circRNAs were derived from 797 genes with diverse functions, including genes involved in the determination of lifespan. A massive accumulation of circRNAs during aging was uncovered. Many hundreds of circRNAs were significantly increased among the aging time-points and increases of select circRNAs by over 40-fold during aging were quantified by RT-qPCR. The expression of 459 circRNAs was determined to be distinct from the expression of linear RNAs from the same host genes, demonstrating host gene independence of circRNA age-accumulation. Conclusions We attribute the global scale of circRNA age-accumulation to the high composition of post-mitotic cells in adult C. elegans, coupled with the high resistance of circRNAs to decay. These findings suggest that the exceptional stability of circRNAs might explain age-accumulation trends observed from neural tissues of other organisms, which also have a high composition of post-mitotic cells. Given the suitability of C. elegans for aging research, it is now poised as an excellent model system to determine whether there are functional consequences of circRNA accumulation during aging. Electronic supplementary material The online version of this article (10.1186/s12864-017-4386-y) contains supplementary material, which is available to authorized users.

Feeding and sleep are highly conserved, interconnected behaviors essential for survival. Starvation has been shown to potently suppress sleep across species; however, whether satiety promotes sleep is still unclear. Here we use the fruit fly, Drosophila melanogaster, as a model organism to address the interaction between feeding and sleep. We first monitored the sleep of flies that had been starved for 24 h and found that sleep amount increased in the first 4 h after flies were given food. Increased sleep after starvation was due to an increase in sleep bout number and average sleep bout length. Mutants of translin or adipokinetic hormone, which fail to suppress sleep during starvation, still exhibited a sleep increase after starvation, suggesting that sleep increase after starvation is not a consequence of sleep loss during starvation. We also found that feeding activity and food consumption were higher in the first 10-30 min after starvation. Restricting food consumption in starved flies to 30 min was sufficient to increase sleep for 1 h. Although flies ingested a comparable amount of food at differing sucrose concentrations, sleep increase after starvation on a lower sucrose concentration was undetectable. Taken together, our results suggest that increased food intake after starvation enhances sleep and reveals a novel relationship between feeding and sleep.

Food and feeding-state dependent changes in chemoreceptor gene expression may allow Caenorhabditis elegans to modify their chemosensory behavior, but the mechanisms essential for these expression changes remain poorly characterized. We had previously shown that expression of a feeding state-dependent chemoreceptor gene, srh-234, in the ADL sensory neuron of C. elegans is regulated via the MEF-2 transcription factor. Here, we show that MEF-2 acts together with basic helix-loop-helix (bHLH) transcription factors to regulate srh-234 expression as a function of feeding state. We identify a cis-regulatory MEF2 binding site that is necessary and sufficient for the starvation-induced down regulation of srh-234 expression, while an E-box site known to bind bHLH factors is required to drive srh-234 expression in ADL. We show that HLH-2 (E/Daughterless), HLH-3 and HLH-4 (Achaete-scute homologs) act in ADL neurons to regulate srh-234 expression. We further demonstrate that the expression levels of srh-234 in ADL neurons are regulated remotely by MXL-3 (Max-like 3 homolog) and HLH-30 (TFEB ortholog) acting in the intestine, which is dependent on insulin signaling functioning specifically in ADL neurons. We also show that this intestine-to-neuron feeding-state regulation of srh-234 involves a subset of insulin-like peptides. These results combined suggest that chemoreceptor gene expression is regulated by both cell-autonomous and non-cell-autonomous transcriptional mechanisms mediated by MEF2 and bHLH factors, which may allow animals to fine-tune their chemosensory responses in response to changes in their feeding state., Author Summary Plasticity in chemoreceptor gene expression may be a simple strategy by which an animal can modulate its chemosensory responses in changing external and internal state conditions. However, the transcriptional mechanisms required for these chemoreceptor gene expression changes are poorly understood. Here, we describe the identification of a transcriptional module(s) consisting of MEF-2 and basic helix-loop-helix (bHLH) transcription factors and their cognate binding sites in Caenorhabditis elegans that act together in ADL sensory neurons to properly regulate expression of a feeding-state dependent chemoreceptor gene. We also showed that chemoreceptor gene expression in ADL neurons are regulated remotely by bHLH factors acting in the intestine through an insulin-mediated signaling pathway, implying a sensory neuron-gut interaction for modulating chemoreceptor gene expression as a function of feeding state. This work describes transcriptional mechanisms mediated by MEF-2 and bHLH factors by which the expression of individual chemoreceptor genes in C. elegans are changed in response to changes in feeding state conditions.

The regulation of chemoreceptor (CR) gene expression by environmental signals and internal cues may contribute to the modulation of multiple physiological processes and behavior in Caenorhabditis elegans. We previously showed that KIN-29, a homolog of salt-inducible kinase, acts in sensory neurons to regulate the expression of a subset of CR genes, as well as sensory behaviors. Here we show that the cGMP-dependent protein kinase EGL-4 acts partly in parallel with KIN-29 to regulate CR gene expression. Sensory inputs inhibit both EGL-4 and KIN-29 functions, and KIN-29 function is inhibited in turn by cAMP-dependent protein kinase (PKA) activation. EGL-4 and KIN-29 regulate CR gene expression by antagonizing the gene repression functions of the class II HDAC HDA-4 and the MEF-2 transcription factor, and KIN-29, EGL-4, and PKA target distinct residues in HDA-4 to regulate its function and subcellular localization. While KIN-29 acts primarily via MEF-2/HDA-4 to regulate additional sensory signal-regulated physiological processes and behaviors, EGL-4 acts via both MEF-2-dependent and -independent pathways. Our results suggest that integration of complex sensory inputs via multiple signaling pathways allows animals to precisely regulate sensory gene expression, thereby appropriately modulating physiology and behavior.

Animals dramatically modify their chemosensory behaviors when starved, which could allow them to alter and optimize their food-search strategies. Dynamic changes in the gene expression of chemoreceptors may be a general mechanism underlying food and state-dependent changes in chemosensory behaviors. In our recent study,(1) we identified chemoreceptors in the ADL sensory neuron type of C. elegans that are modulated by feeding state and food availability. Here, we highllight our recent findings by which sensory inputs into ADL, neuronal outputs from ADL, and circuit inputs from the RMG interneuron, which is electrically connected to ADL, are required to regulate an ADL-expressed chemoreceptor. This sensory and circuit-mediated regulation of chemoreceptor gene expression is dependent on cell-autonomous pathways acting in ADL, e.g. KIN-29, DAF-2, OCR-2 and calcium signaling, and circuit inputs from RMG mediated by NPR-1. Based on these findings, we propose an intriguing but speculative feedback modulatory circuit mechanism by which sensory perception of food and internal state signals may be coupled to regulate ADL-expressed chemoreceptors, which may allow animals to precisely regulate and fine-tune their chemosensory neuron responses as a function of feeding state.

Background Locomotor activity is used extensively as a behavioral output to study the underpinnings of circadian rhythms. Recent studies have required a populational approach for the study of circadian rhythmicity in Caenorhabditis elegans locomotion. New method We describe an imaging system for long-term automated recording and analysis of locomotion data of multiple free-crawling C. elegans animals on the surface of an agar plate. We devised image analysis tools for measuring specific features related to movement and shape to identify circadian patterns. Results We demonstrate the utility of our system by quantifying circadian locomotor rhythms in wild-type and mutant animals induced by temperature cycles. We show that 13 °C:18 °C (12:12 h) cycles are sufficient to entrain locomotor activity of wild-type animals, which persist but are rapidly damped during 13 °C free-running conditions. Animals with mutations in tax-2 , a cyclic nucleotide-gated (CNG) ion channel, significantly reduce locomotor activity during entrainment and free-running. Comparison with existing method(s) Current methods for measuring circadian locomotor activity is generally restricted to recording individual swimming animals of C. elegans , which is a distinct form of locomotion from crawling behavior generally observed in the laboratory. Our system works well with up to 20 crawling adult animals, and allows for a detailed analysis of locomotor activity over long periods of time. Conclusions Our population-based approach provides a powerful tool for quantification of circadian rhythmicity of C. elegans locomotion, and could allow for a screening system of candidate circadian genes in this model organism.

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting with Caenorhabditis elegans Gα subunits. The genome of C. elegans contains homologues of each of the four mammalian classes of Gα subunits (Gs, Gi/o, Gq and G12), and 17 other Gα subunits. We tested 19 of the GGα subunits and four constitutively activated Gα subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Gα subunits. This set includes C. elegans orthologues of known interactors of Gα subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Gα subunit but variable for the activation status of the Gα subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal.

The G(12) type of heterotrimeric G-proteins play an important role in development and behave as potent oncogenes in cultured cells. However, little is known about the molecular nature of the components that act in the G(12)-signaling pathway in an organism. We characterized a C. elegans Galpha subunit gene, gpa-12, which is a homolog of mammalian G(12)/G(13)alpha, and found that animals defective in gpa-12 are viable. Expression of activated GPA-12 (G(12)QL) results in a developmental growth arrest caused by a feeding behavior defect that is due to a dramatic reduction in pharyngeal pumping. To elucidate the molecular nature of the signaling pathways in which G(12) participates, we screened for suppressors of the G(12)QL phenotype. We isolated 50 suppressors that contain mutations in tpa-1, which encodes two protein kinase C isoforms, TPA-1A and TPA-1B, most similar to PKCtheta/delta. TPA-1 mediates the action of the tumor promoter PMA. Expression of G(12)QL and treatment of wild-type animals with PMA induce an identical growth arrest caused by inhibition of larval feeding, which is dependent on TPA-1A and TPA-1B function. These results suggest that TPA-1 is a downstream target of both G(12) signaling and PMA in modulating feeding and growth in C. elegans. Taken together, our findings provide a potential molecular mechanism for the transforming capability of G(12) proteins.

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting withCaenorhabditis elegansGα subunits. The genome ofC. eleganscontains homologues of each of the four mammalian classes of Gα subunits (Gs, Gi/o, Gq and G12), and 17 other Gα subunits. We tested 19 of the GGα subunits and four constitutively activated Gα subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Gα subunits. This set includesC. elegansorthologues of known interactors of Gα subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Gα subunit but variable for the activation status of the Gα subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal.

Feeding state and food availability can dramatically alter an animals' sensory response to chemicals in its environment. Dynamic changes in the expression of chemoreceptor genes may underlie some of these food and state-dependent changes in chemosensory behavior, but the mechanisms underlying these expression changes are unknown. Here, we identified a KIN-29 (SIK)-dependent chemoreceptor, srh-234, in C. elegans whose expression in the ADL sensory neuron type is regulated by integration of sensory and internal feeding state signals. We show that in addition to KIN-29, signaling is mediated by the DAF-2 insulin-like receptor, OCR-2 TRPV channel, and NPR-1 neuropeptide receptor. Cell-specific rescue experiments suggest that DAF-2 and OCR-2 act in ADL, while NPR-1 acts in the RMG interneurons. NPR-1-mediated regulation of srh-234 is dependent on gap-junctions, implying that circuit inputs regulate the expression of chemoreceptor genes in sensory neurons. Using physical and genetic manipulation of ADL neurons, we show that sensory inputs from food presence and ADL neural output regulate srh-234 expression. While KIN-29 and DAF-2 act primarily via the MEF-2 (MEF2) and DAF-16 (FOXO) transcription factors to regulate srh-234 expression in ADL neurons, OCR-2 and NPR-1 likely act via a calcium-dependent but MEF-2- and DAF-16-independent pathway. Together, our results suggest that sensory- and circuit-mediated regulation of chemoreceptor genes via multiple pathways may allow animals to precisely regulate and fine-tune their chemosensory responses as a function of internal and external conditions., Author Summary Animals dramatically modify their chemosensory behaviors to attractive and noxious chemical stimuli when starved. This could allow them to alter and optimize their food-search strategies to increase their survival and reproduction. Changes in the gene expression of chemoreceptors specialized in detecting environmental stimuli is observed in fish, insects and nematodes, and may be a general mechanism underlying the changes in chemosensory behaviors observed in starved animals. To elucidate this mechanism, we have developed an in vivo reporter assay in C. elegans for monitoring the expression of a candidate chemoreceptor gene in a single sensory neuron type, called ADL, as a function of feeding state. Using this reporter assay, we show that sensory inputs into ADL and neural outputs from ADL, as well as inputs from the RMG interneuron, which is electrically connected to ADL, are required to fine-tune expression of chemoreceptor genes in ADL. Sensory and circuit-mediated regulation of chemoreceptor gene expression is dependent on multiple pathways, including the neuropeptide receptor, NPR-1, and the DAF-2 insulin-like receptor. Our results reveal mechanisms underlying chemoreceptor gene expression, and provide insight into how expression changes in chemoreceptor genes may contribute to changes in chemosensory behavior as a function of feeding state.

Most organisms have an endogenous circadian clock that is synchronized to environmental signals such as light and temperature. Although circadian rhythms have been described in the nematode Caenorhabditis elegans at the behavioral level, these rhythms appear to be relatively non-robust. Moreover, in contrast to other animal models, no circadian transcriptional rhythms have been identified. Thus, whether this organism contains a bona fide circadian clock remains an open question. Here we use genome-wide expression profiling experiments to identify light- and temperature-entrained oscillating transcripts in C. elegans. These transcripts exhibit rhythmic expression with temperature-compensated 24-h periods. In addition, their expression is sustained under constant conditions, suggesting that they are under circadian regulation. Light and temperature cycles strongly drive gene expression and appear to entrain largely nonoverlapping gene sets. We show that mutations in a cyclic nucleotide-gated channel required for sensory transduction abolish both light- and temperature-entrained gene expression, implying that environmental cues act cell nonautonomously to entrain circadian rhythms. Together, these findings demonstrate circadian-regulated transcriptional rhythms in C. elegans and suggest that further analyses in this organism will provide new information about the evolution and function of this biological clock.

The generation of cellular diversity is dependent on the precise spatiotemporal regulation of gene expression by both cis- and trans-acting mechanisms. The developmental principles regulating expression of specific gene subsets in individual cell types are not fully understood. Here we define the cis-regulatory mechanisms driving expression of cell-selective and broadly expressed genes in vivo in the AWB olfactory neuron subtype in C. elegans. We identify an element that is necessary to drive expression of neuron-selective chemoreceptor genes in the AWB neurons, and show that this element functions in a context-dependent manner. We find that the expression of broadly expressed sensory neuronal genes in the AWB neurons is regulated by diverse cis- and trans-regulatory mechanisms that act partly in parallel to the pathways governing expression of AWB-selective genes. We further demonstrate that cis-acting mechanisms driving gene expression in the AWB neurons appear to have diverged in related nematode species. Our results provide insights into the cis-regulatory logic driving cell-specific gene expression, and suggest that variations in this logic contribute to the generation of functional diversity. Developmental Dynamics 238:3080–3092, 2009. © 2009 Wiley-Liss, Inc.

To identify novel components in heterotrimeric G-protein signalling, we performed an extensive screen for proteins interacting with Caenorhabditis elegans Galpha subunits. The genome of C. elegans contains homologues of each of the four mammalian classes of Galpha subunits (Gs, Gi/o, Gq and G12), and 17 other Galpha subunits. We tested 19 of the GGalpha subunits and four constitutively activated Galpha subunits in a largescale yeast two-hybrid experiment. This resulted in the identification of 24 clones, representing 11 different proteins that interact with four different Galpha subunits. This set includes C. elegans orthologues of known interactors of Galpha subunits, such as AGS3 (LGN/PINS), CalNuc and Rap1Gap, but also novel proteins, including two members of the nuclear receptor super family and a homologue of human haspin (germ cell-specific kinase). All interactions were found to be unique for a specific Galpha subunit but variable for the activation status of the Galpha subunit. We used expression pattern and RNA interference analysis of the G-protein interactors in an attempt to substantiate the biological relevance of the observed interactions. Furthermore, by means of a membrane recruitment assay, we found evidence that GPA-7 and the nuclear receptor NHR-22 can interact in the animal.

Background The left and right AWC olfactory neurons in Caenorhabditis elegans differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWCOFF, and the contralateral AWC becomes AWCON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWCON. Results We show here that the asymmetry gene olrn-1 acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWCON. OLRN-1, a protein with potential membrane-association domains, is related to the Drosophila Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. olrn-1 opposes the action of two voltage-activated calcium channel homologs, unc-2 (CaV2) and egl-19 (CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWCOFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from olrn-1. Conclusion olrn-1 and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.

The expression of individual chemoreceptor (CR) genes in Caenorhabditis elegans is regulated by multiple environmental and developmental cues, possibly enabling C. elegans to modulate its sensory responses. We had previously shown that KIN-29, a member of the salt-inducible kinase family, acts in a subset of chemosensory neurons to regulate the expression of CR genes, body size and entry into the alternate dauer developmental stage. Here, we show that KIN-29 regulates these processes by phosphorylating the HDA-4 class II histone deacetylase (HDAC) and inhibiting the gene repression functions of HDA-4 and an MEF-2 MADS domain transcription factor. MEF-2 binds directly to the CR gene regulatory sequences, and is required only to repress but not activate CR gene expression. A calcineurin phosphatase antagonizes the KIN-29/MEF-2-regulated pathway to modulate levels of CR gene expression. Our results identify KIN-29 as a new regulator of MEF2/HDAC functions in the nervous system, reveal cell-specific mechanisms of action of this pathway in vivo and demonstrate remarkable complexity in the regulation of CR gene expression in C. elegans.

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms., The screen suggested functions for 393 genes for which no RNAi-mediated phenotype was known. The comparison with similar screens in worms has general implications for RNAi experiments

Expression of a constitutively activated version of the heterotrimeric G protein alpha-subunit Galphas results in the swelling and vacuolization of a specific subset of ventral nerve cord motoneurons of Caenorhabditis elegans. A second site modifier (sgs-1) that completely suppresses this neuronal degeneration has been isolated. sgs-1 was cloned and was shown to encode an adenylyl cyclase which is most similar to mammalian adenylyl cyclase type IX. Mutations in sgs-1 change residues that are conserved among different adenylyl cyclases. These mutations are located in the two catalytic domains and in the first multiple transmembrane spanning region of the predicted protein. An sgs-1 reporter construct shows a general neuronal expression pattern, demonstrating that sgs-1 is expressed in the neurons that are susceptible to activated Galphas-induced cell death. A second C.elegans adenylyl cyclase gene (acy-2) was analyzed as well. In contrast to sgs-1, acy-2 shows a restricted expression pattern and loss of acy-2 function results in early larval lethality. These results suggest that SGS-1 is a target of Galphas signaling in motoneurons, whereas an interaction of Galphas with ACY-2, probably in the canal-associated neurons, is required for viability.

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation., Synopsis The authors have utilized Caenorhabditis elegans, and yeast genetics, combined with mammalian tissue and cell culture experiments to investigate the mechanism of action of a unique set of small molecules. These molecules are active in tissue models of urinary incontinence and allow for increased bladder filling. In the course of studying sensitivity and resistance to these compounds, Fitzgerald et al. uncovered novel alleles of RGS and Gq proteins. Further characterization of one such allele identified that its action conferred a hypo-adaptive phenotype on yeast during pheromone signaling assays. Their data as a whole indicate that these small molecules are able to diminish signaling from G-protein coupled receptors (GPCR) downstream of the receptors themselves. Since GPCR signaling is very important in many diseases in humans, the novel mechanism of these compounds may offer new ways to treat human disease.