Deletion of smn-1, the Caenorhabditis elegans ortholog of the spinal muscular atrophy gene, results in locomotor dysfunction and reduced lifespan.

Spinal muscular atrophy is the most common genetic cause of infant mortality and is characterized by degeneration of lower motor neurons leading to muscle wasting. The causative gene has been identified as survival motor neuron (SMN). The invertebrate model organism Caenorhabditis elegans contains smn-1, the ortholog of human SMN. Caenorhabditis elegans smn-1 is expressed in various tissues including the nervous system and body wall muscle, and knockdown of smn-1 by RNA interference is embryonic lethal. Here we show that the smn-1(ok355) deletion, which removes most of smn-1 including the translation start site, produces a pleiotropic phenotype including late larval arrest, reduced lifespan, sterility as well as impaired locomotion and pharyngeal activity. Mutant nematodes develop to late larval stages due to maternal contribution of the smn-1 gene product that allows to study SMN-1 functions beyond embryogenesis. Neuronal, but not muscle-directed, expression of smn-1 partially rescues the smn-1(ok355) phenotype. Thus, the deletion mutant smn-1(ok355) provides a useful platform for functional analysis of an invertebrate ortholog of the human SMN protein.

Caenorhabditis elegans in the study of SMN-interacting proteins: a role for SMI-1, an orthologue of human Gemin2 and the identification of novel components of the SMN complex.

Spinal muscular atrophy is a common neuromuscular disorder caused by mutations in the survival motor neuron (SMN) gene. In mammals, SMN is tightly associated with Gemin2. To gain further insight into the functions of SMN and Gemin2, we have cloned and sequenced smi-1 (Survival of Motor neuron-Interacting protein 1), a C. elegans homologue of the human Gemin2 gene. We show that the SMI-1 expression pattern and RNA interference phenotype show considerable overlap with that previously reported for SMN-1. Finally, we demonstrate that the SMN-1 and SMI-1 proteins directly interact. Having demonstrated the utility of the C. elegans genetic model for investigating genes encoding SMN-interacting proteins, we have undertaken a yeast two-hybrid screen of a C. elegans cDNA library to identify novel proteins that interact with SMN-1. We show the direct interaction of SMN-1 with nine novel proteins, several of which may be involved in RNA metabolism.

Alpha7 mutants mimicking atypical motifs (YxxCC of loop-C, and E to H at -1' in TM2) in the C. elegans LEV-8 subunit affect nicotinic acetylcholine receptor function.

The ACR-8-like group of C. elegans nicotinic acetylcholine receptor (nAChR) subunits contain unusual motifs in the ACh binding site and in the -1' position of transmembrane region two (TM2). Using site-directed mutagenesis (SDM) we have introduced these motifs into chicken alpha7 as it has not been possible to express C. elegans nAChR in vitro. Oocytes expressing alpha7 with the C. elegans binding motif show a reduced affinity and efficacy for both ACh and nicotine. The blocking action of the anthelmintic drug levamisole is reduced. The TM2 motif resulted in a non-functional receptor. We conclude that the TM2 motif profoundly restricts cation movement through the alpha7 channel but does not confer anion permeability. The altered form of the ACh binding motif is likely to result in a receptor with altered pharmacology, adding potential functional diversity at synapses in the nervous system and neuromuscular junctions of C. elegans.

Gene silencing of selected calcium-signalling molecules in a Drosophila cell line using double-stranded RNA interference.

Using the Drosophila melanogaster S2 cell line, stably expressing a cloned muscarinic acetylcholine receptor (AChR), DM1, we have applied gene silencing by double-stranded RNA interference (RNAi) to knock down gene products involved in DM1-mediated calcium signalling. We have shown that RNAi knock down of either the inositol 1,4,5-trisphosphate receptor (Ins(1,4,5)P(3)R), or the SERCA calcium pump in the S2-DM1 cells blocks the increase in intracellular calcium concentration ([Ca(2+)](i)) resulting from activation of the DM1 receptor by 100 microM carbamylcholine (CCh). When RNAi designed to knock down the ryanodine receptor (RyR) was tested, there was no change in the calcium increase detected in response to CCh, consistent with a failure to detect RyRs in S2-DM1 cells using RT-PCR. A combination of RNAi and calcium imaging has provided a direct demonstration of key roles for the Ins(1,4,5)P(3)R and the SERCA pump in the response to DM1 receptor activation.Thus, we show that silencing of individual genes by RNAi in a well characterised Drosophila S2 cell line offers experimental opportunities for cell-signalling studies. Future investigations with RNAi libraries taking full advantage of the wealth of new information available from sequencing the Drosophila genome, may help identify novel components of cell-signalling pathways and functionally linked gene products.

Gene expression profiling studies on Caenorhabditis elegans dystrophin mutants dys-1(cx-35) and dys-1(cx18).

The Caenorhabditis elegans genome contains a single dystrophin/utrophin orthologue, dys-1. Point mutations in this gene, dys-1(cx35) and dys-1(cx18), result in truncated proteins. Such mutants offer potentially valuable worm models of human Duchenne muscular dystrophy. We have used microarrays to examine genes expressed differentially between wild-type C. elegans and dys-1 mutants. We found 106 genes (115 probe sets) to be differentially expressed when the two mutants are compared to wild-type worms, 49 of which have been assigned to six functional categories. The main categories of regulated genes in C. elegans are genes encoding intracellular signalling, cell-cell communication, cell-surface, and extracellular matrix proteins; genes in these same categories have been shown by others to be differentially expressed in muscle biopsies of muscular dystrophy patients. The C. elegans model may serve as a convenient vehicle for future genetic and chemical screens to search for new drug targets.

The Caenorhabditis elegans lev-8 gene encodes a novel type of nicotinic acetylcholine receptor alpha subunit.

We have cloned Caenorhabditis elegans lev-8 and demonstrated that it encodes a novel nicotinic acetylcholine receptor (nAChR) subunit (previously designated ACR-13), which has functional roles in body wall and uterine muscles as part of a levamisole-sensitive receptor. LEV-8 is an alpha subunit and is the first to be described from the ACR-8-like group, a new class of nAChR with atypical acetylcholine-binding site (loop C) and channel-lining motifs. A single base pair change in the first intron of lev-8 in lev-8(x15) mutants leads to alternative splicing and the introduction of a premature stop codon. lev-8(x15) worms are partially resistant to levamisole-induced egg laying and paralysis, phenotypes rescued by expression of the wild-type gene. lev-8(x15) worms also show reduced rates of pharyngeal pumping. Electrophysiological recordings from body wall muscle show that currents recorded in response to levamisole have reduced amplitude in lev-8(x15) compared with wild-type animals. Consistent with these phenotypic observations, green fluorescent protein fused to LEV-8 is expressed in body wall and uterine muscle, motor neurons and epithelial-derived socket cells. Thus, LEV-8 is a levamisole receptor subunit and exhibits the most diverse expression pattern of any invertebrate nAChR subunit studied to date.

A Drosophila melanogaster cell line (S2) facilitates post-genome functional analysis of receptors and ion channels.

The complete sequencing of the genome of the fruit fly Drosophila melanogaster offers the prospect of detailed functional analysis of the extensive gene families in this genetic model organism. Comprehensive functional analysis of family members is facilitated by access to a robust, stable and inducible expression system in a fly cell line. Here we show how the Schneider S2 cell line, derived from the Drosophila embryo, provides such an expression system, with the bonus that radioligand binding studies, second messenger assays, ion imaging, patch-clamp electrophysiology and gene silencing can readily be applied. Drosophila is also ideal for the study of new control strategies for insect pests since the receptors and ion channels that many new animal health drugs and crop protection chemicals target can be expressed in this cell line. In addition, many useful orthologues of human disease genes are emerging from the Drosophila genome and the study of their functions and interactions is another area for postgenome applications of S2 cell lines.