Alexander M. van der Linden

The C. elegans SIK3 homolog KIN-29 regulates the interaction between sleep and metabolism, but mechanisms underlying this regulation are not understood. Here, we show that KIN-29 regulates sleep that is induced by cellular stress (stress-induced sleep or SIS) through mitochondrial reactive oxygen species (ROS) signaling. Following sleep-promoting ultraviolet-C (UVC) irradiation, mitochondrial ROS rises in concert with sleep in wild-type but not in sleepless kin-29 mutants. k kin-29 mutants have reduced mitochondrial ROS, reduced oxygen consumption rates, and are resistant to oxidative stress. Transcriptomic and proteomic profiling of kin-29 mutants reveal enrichment for genes involved in ROS mitigation such as the mitochondrial superoxide dismutase SOD-3. Consistent with the notion that ROS promotes sleep, genetic disruption of mitochondrial SODs enhances UVC-induced SIS. Increasing mitochondrial ROS using the optogenetic tool SuperNova partially restores SIS in kin-29 mutants but not in mutants with defective function of the sleep-inducing ALA neuron; this suggests that mitochondrial ROS act downstream of kin-29 but upstream of ALA. The identification of mitochondrial ROS as a nematode sleep regulator supports a phylogenetically conserved mechanism by which metabolic stress and SIKs promote sleep.

Circular RNA (circRNA) biogenesis is regulated by RNA-binding proteins (RBPs) that alter back-splicing of exons in protein-coding genes. However, few in vivo roles for RBPs in the regulation of circRNA biogenesis have been characterized. We previously showed that many circRNAs increase with age in Caenorhabditis elegans, and that loss of circ-crh-1, an abundant age-accumulated circRNA, extends mean lifespan. Given the established role of the mammalian RBP NOVA2 in promoting circRNA biogenesis, we investigated whether nova-1, the sole C. elegans homolog of NOVA1/2, similarly regulates circRNA expression and function in vivo. RNA-sequencing of nova-1 mutants compared to wild-type identified 686 circRNAs. Of these, 103 were differentially expressed in nova-1 mutants compared to wild-type, with 76 upregulated and 27 downregulated circRNAs, suggesting NOVA-1 acts as a negative regulator of a subset of circRNAs. nova-1 mutants also exhibited linear alternative splicing changes, primarily in alternative 3′ splice site usage and exon skipping, and showed minimal overlap with circRNA loci. Notably, circ-crh-1 represented a shared regulatory target, suggesting NOVA-1 may coordinate splicing regulation with the production of crh-1 circRNAs. Motif analysis further revealed that over half of the NOVA-1-regulated splicing events contained YCAY motif sites, with crh-1 harboring a high density of sites, consistent with its alternative 3′ splice site usage and circRNA production. Finally, nova-1 mutants exhibited an extended mean lifespan and enhanced heat stress recovery. Together, these findings identify NOVA-1 as a key regulator of circRNA expression and alternative splicing in C. elegans, with likely downstream consequences for organismal lifespan and stress resilience.

Circular RNAs (circRNAs) are non-coding RNAs mostly derived from exons of protein-coding genes via a back-splicing process. The expression of hundreds of circRNAs accumulates during healthy aging and is associated with Alzheimer’s disease (AD), which is characterized by the accumulation of amyloid-beta (Aβ) proteins. In C. elegans, many circRNAs were previously found to accumulate during aging, with loss of age-accumulated circRNAs derived from the CREB gene (circ-crh-1) to increase mean lifespan. Here, we used C. elegans to study the effects of age-accumulated circRNAs on the age-related onset of Aβ-toxicity. We found that circ-crh-1 mutations delayed Aβ-induced muscle paralysis and lifespan phenotypes in a transgenic C. elegans strain expressing a full-length human Aβ-peptide (Aβ_1–42) selectively in muscle cells (GMC101). The delayed Aβ phenotypic defects were associated with the inhibition of Aβ aggregate deposition, and thus, genetic removal of circ-crh-1 alleviated Aβ-induced toxicity. Consistent with a detrimental role for age-accumulated circRNAs in AD, the expression level of circ-crh-1 expression is elevated after induction of Aβ during aging, whereas linear crh-1 mRNA expression remains unchanged. Finally, we found that the delayed onset of Aβ-induced paralysis observed in circ-crh-1 mutants is dependent on the col-49 collagen gene. Taken together, our results show that the loss of an age-accumulated circRNA exerts a protective role on Aβ-induced toxicity, demonstrating the utility of C. elegans for studying circRNAs in AD and its relationship to aging.

Circular RNAs (circRNAs) are non-coding RNAs mostly derived from exons of protein-coding genes via a back-splicing process. The expression of hundreds of circRNAs accumulates during healthy aging and is associated with Alzheimer’s disease (AD), characterized by the accumulation of amyloid-beta (Aβ) proteins. In C. elegans , many circRNAs were previously found to accumulate during aging, with loss of age-accumulated circRNAs derived from the CREB gene (circ- crh-1 ) to increase mean lifespan. Here, we used C. elegans to study the effects of age-accumulated circRNAs on the age-related onset of Aβ-toxicity. We found that circ- crh-1 mutations delayed Aβ-induced muscle paralysis and lifespan phenotypes in a transgenic C. elegans strain expressing a full-length human Aβ-peptide (Aβ _1-42 ) selectively in muscle cells (GMC101). The delayed Aβ phenotypic defects were associated with inhibiting the deposition of Aβ aggregates, and thus, genetic removal of circ- crh-1 provides protection against Aβ-induced toxicity. Consistent with a detrimental role for age-accumulated circRNAs in AD, circ- crh-1 expression level is elevated after induction of Aβ during aging, whereas linear crh-1 mRNA expression remains unchanged. Finally, we show that a circ- crh-1 upregulated collagen gene, col-49 , promotes Aβ-induced paralysis. Taken together, our results show that the loss of an age-accumulated circRNA exerts a protective role on Aβ-induced toxicity, demonstrating the utility of C. elegans for studying circRNAs in AD and its relationship to aging.

Dynamic changes in chemoreceptor gene expression levels in sensory neurons are 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 Caenorhabditis elegans is downregulated when animals are fed a Comamonas aquatica bacterial diet, but not on an Escherichia coli diet. Remarkably, this diet-modulated effect on srh-234 expression is dependent on the micronutrient vitamin B12 endogenously produced by Comamonas aq. bacteria. Excess propionate and genetic perturbations in the canonical and shunt propionate breakdown pathways are able to override the repressive effects of vitamin B12 on srh-234 expression. The vitamin B12-mediated regulation of srh-234 expression levels in ADL requires the MEF-2 MADS domain 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.

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.

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.

Many lines of evidence point to links between sleep regulation and energy homeostasis, but mechanisms underlying these connections are unknown. During C. elegans sleep, energetic stores are allocated to non-neural 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 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 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. Highlights: Sleep is associated with fat mobilization and low ATP levels Metabolic regulation of sleep requires the salt-induced kinase (SIK) homolog KIN-29 KIN-29 acts in sensory neurons upstream of sleep-promoting neurons Nuclear localization of KIN-29 is required for the metabolic regulation of sleep A 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.