MUT-7 Provides Molecular Insight into the Werner Syndrome Exonuclease.

Werner syndrome (WS) is a rare recessive genetic disease characterized by premature aging. Individuals with this disorder develop normally during childhood, but their physiological conditions exacerbate the aging process in late adolescence. WS is caused by mutation of the human WS gene (WRN), which encodes two main domains, a 3'-5' exonuclease and a 3'-5' helicase. Caenorhabditis elegans expresses human WRN orthologs as two different proteins: MUT-7, which has a 3'-5' exonuclease domain, and C. elegans WRN-1 (CeWRN-1), which has only helicase domains. These unique proteins dynamically regulate olfactory memory in C. elegans, providing insight into the molecular roles of WRN domains in humans. In this review, we specifically focus on characterizing the function of MUT-7 in small interfering RNA (siRNA) synthesis in the cytoplasm and the roles of siRNA in directing nuclear CeWRN-1 loading onto a heterochromatin complex to induce negative feedback regulation. Further studies on the different contributions of the 3'-5' exonuclease and helicase domains in the molecular mechanism will provide clues to the accelerated aging processes in WS.

C. elegans orthologs MUT-7/CeWRN-1 of Werner syndrome protein regulate neuronal plasticity.

Caenorhabditis elegans expresses human Werner syndrome protein (WRN) orthologs as two distinct proteins: MUT-7, with a 3'-5' exonuclease domain, and CeWRN-1, with helicase domains. How these domains cooperate remains unclear. Here, we demonstrate the different contributions of MUT-7 and CeWRN-1 to 22G small interfering RNA (siRNA) synthesis and the plasticity of neuronal signaling. MUT-7 acts specifically in the cytoplasm to promote siRNA biogenesis and in the nucleus to associate with CeWRN-1. The import of siRNA by the nuclear Argonaute NRDE-3 promotes the loading of the heterochromatin-binding protein HP1 homolog HPL-2 onto specific loci. This heterochromatin complex represses the gene expression of the guanylyl cyclase ODR-1 to direct olfactory plasticity in C. elegans. Our findings suggest that the exonuclease and helicase domains of human WRN may act in concert to promote RNA-dependent loading into a heterochromatin complex, and the failure of this entire process reduces plasticity in postmitotic neurons.

Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides.

Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 µg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies.

Expression of an expanded CGG-repeat RNA in a single pair of primary sensory neurons impairs olfactory adaptation in Caenorhabditis elegans.

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a severe neurodegenerative disorder that affects carriers of premutation CGG-repeat expansion alleles of the fragile X mental retardation 1 (FMR1) gene; current evidence supports a causal role of the expanded CGG repeat within the FMR1 mRNA in the pathogenesis of FXTAS. Though the mRNA has been observed to induce cellular toxicity in FXTAS, the mechanisms are unclear. One common neurophysiological characteristic of FXTAS patients is their inability to properly attenuate their response to an auditory stimulus upon receipt of a small pre-stimulus. Therefore, to gain genetic and cell biological insight into FXTAS, we examined the effect of expanded CGG repeats on the plasticity of the olfactory response of the genetically tractable nematode, Caenorhabditis elegans (C. elegans). While C. elegans is innately attracted to odors, this response can be downregulated if the odor is paired with starvation. We found that expressing expanded CGG repeats in olfactory neurons interfered with this plasticity without affecting either the innate odor-seeking response or the olfactory neuronal morphology. Interrogation of three RNA regulatory pathways indicated that the expanded CGG repeats act via the C. elegans microRNA (miRNA)-specific Argonaute ALG-2 to diminish olfactory plasticity. This observation suggests that the miRNA-Argonaute pathway may play a pathogenic role in subverting neuronal function in FXTAS.

Endogenous nuclear RNAi mediates behavioral adaptation to odor.

Most eukaryotic cells express small regulatory RNAs. The purpose of one class, the somatic endogenous siRNAs (endo-siRNAs), remains unclear. Here, we show that the endo-siRNA pathway promotes odor adaptation in C. elegans AWC olfactory neurons. In adaptation, the nuclear Argonaute NRDE-3, which acts in AWC, is loaded with siRNAs targeting odr-1, a gene whose downregulation is required for adaptation. Concomitant with increased odr-1 siRNA in AWC, we observe increased binding of the HP1 homolog HPL-2 at the odr-1 locus in AWC and reduced odr-1 mRNA in adapted animals. Phosphorylation of HPL-2, an in vitro substrate of the EGL-4 kinase that promotes adaption, is necessary and sufficient for behavioral adaptation. Thus, environmental stimulation amplifies an endo-siRNA negative feedback loop to dynamically repress cognate gene expression and shape behavior. This class of siRNA may act broadly as a rheostat allowing prolonged stimulation to dampen gene expression and promote cellular memory formation. PAPERFLICK:

The C. elegans cGMP-dependent protein kinase EGL-4 regulates nociceptive behavioral sensitivity.

Signaling levels within sensory neurons must be tightly regulated to allow cells to integrate information from multiple signaling inputs and to respond to new stimuli. Herein we report a new role for the cGMP-dependent protein kinase EGL-4 in the negative regulation of G protein-coupled nociceptive chemosensory signaling. C. elegans lacking EGL-4 function are hypersensitive in their behavioral response to low concentrations of the bitter tastant quinine and exhibit an elevated calcium flux in the ASH sensory neurons in response to quinine. We provide the first direct evidence for cGMP/PKG function in ASH and propose that ODR-1, GCY-27, GCY-33 and GCY-34 act in a non-cell-autonomous manner to provide cGMP for EGL-4 function in ASH. Our data suggest that activated EGL-4 dampens quinine sensitivity via phosphorylation and activation of the regulator of G protein signaling (RGS) proteins RGS-2 and RGS-3, which in turn downregulate Gα signaling and behavioral sensitivity.

Nuclear entry of a cGMP-dependent kinase converts transient into long-lasting olfactory adaptation.

To navigate a complex and changing environment, an animal's sensory neurons must continually adapt to persistent cues while remaining responsive to novel stimuli. Long-term exposure to an inherently attractive odor causes Caenorhabditis elegans to ignore that odor, a process termed odor adaptation. Odor adaptation is likely to begin within the sensory neuron, because it requires factors that act within these cells at the time of odor exposure. The process by which an olfactory sensory neuron makes a decisive shift over time from a receptive state to a lasting unresponsive one remains obscure. In C. elegans, adaptation to odors sensed by the AWC pair of olfactory neurons requires the cGMP-dependent protein kinase EGL-4. Using a fully functional, GFP-tagged EGL-4, we show here that prolonged odor exposure sends EGL-4 into the nucleus of the stimulated AWC neuron. This odor-induced nuclear translocation correlates temporally with the stable dampening of chemotaxis that is indicative of long-term adaptation. Long-term adaptation requires cGMP binding residues as well as an active EGL-4 kinase. We show here that EGL-4 nuclear accumulation is both necessary and sufficient to induce long-lasting odor adaptation. After it is in the AWC nucleus, EGL-4 decreases the animal's responsiveness to AWC-sensed odors by acting downstream of the primary sensory transduction. Thus, the EGL-4 protein kinase acts as a sensor that integrates odor signaling over time, and its nuclear translocation is an instructive switch that allows the animal to ignore persistent odors.

Purification and characterization of the Caenorhabditis elegans HCF protein and domains of human HCF.

The human cellular factor (HCF) is a multidomain protein that is implicated in processes of cell cycle progression, and it is recruited into a multicomponent assembly that triggers the expression of the herpes simplex virus genome. The amino-terminal domain of HCF has been proposed to form a "kelch" type beta-propeller fold, and the carboxy-terminal domain contains a repeat of a fibronectin-like motif. We describe the expression, purification, and characterization of the domains from the human HCF and of the full-length HCF from Caenorhabditis elegans. The purified recombinant C. elegans HCF can substitute for the human HCF in efficiently forming a multiprotein complex on a herpes simplex virus promoter element. As noted in earlier studies, a segment of human HCF encompassing the human kelch domain forms a stable complex on a viral promoter element. The purified fibronectin domain can also be recruited into this complex, but not into the stable complex formed with the minimal kelch domain. These results suggest that the fibronectin domain can interact with HCF in the transcriptional activating complex and that the association requires a region outside the putative beta-propeller.