The nematode cuticle is an extremely flexible and resilient exoskeleton that permits locomotion via attachment to muscle, confers environmental protection and allows growth by molting. It is synthesised five times, once in the embryo and subsequently at the end of each larval stage prior to molting. It is a highly structured extra-cellular matrix (ECM), composed predominantly of cross-linked collagens, additional insoluble proteins termed cuticlins, associated glycoproteins and lipids. The cuticle collagens are encoded by a large gene family that are subject to strict patterns of temporal regulation. Cuticle collagen biosynthesis involves numerous co- and post-translational modification, processing, secretion and cross-linking steps that in turn are catalysed by specific enzymes and chaperones. Mutations in individual collagen genes and their biosynthetic pathway components can result in a range of defects from abnormal morphology (dumpy and blister) to embryonic and larval death, confirming an essential role for this structure and highlighting its potential as an ECM experimental model system.

The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons (PTCs). Seven genes (smg-1-7, for suppressor with morphological effect on genitalia) that are essential for NMD were originally identified in the nematode Caenorhabditis elegans, and orthologs of these genes have been found in several species. Whereas in humans NMD is linked to splicing, PTC definition occurs independently of exon boundaries in Drosophila. Here, we have conducted an analysis of the cis-acting sequences and trans-acting factors that are required for NMD in C. elegans. We show that a PTC codon is defined independently of introns in C. elegans and, consequently, components of the exon junction complex (EJC) are dispensable for NMD. We also show a distance-dependent effect, whereby PTCs that are closer to the 3 end of the mRNA are less sensitive to NMD. We also provide evidence for the existence of previously unidentified components of the NMD pathway that, unlike known smg genes, are essential for viability in C. elegans. A genome-wide RNA interference (RNAi) screen resulted in the identification of two such novel NMD genes, which are essential for proper embryonic development, and as such represent a new class of essential NMD genes in C. elegans that we have termed smgl (for smg lethal). We show that the encoded proteins are conserved throughout evolution and are required for NMD in C. elegans and also in human cells.[Keywords: Nonsense-mediated decay; smg genes; RNAi screens; exon junction complex; C. elegans] Supplemental material is available at http://www.genesdev.org. Nonsense-mediated mRNA decay (NMD) is a highly conserved surveillance mechanism present in all eukaryotes examined that prevents the synthesis of truncated proteins. It does so by promoting the degradation of mRNAs containing premature translation termination codons (PTCs) (for a recent review, see Maquat 2004). The functional importance of NMD is highlighted by the fact that this pathway targets for degradation a wide array of endogenous transcripts, as well as erroneously processed transcripts and transcripts carrying spontaneous mutations (for review, see Rehwinkel et al. 2006). It is proposed that NMD modulates the phenotypic outcome of many diseases, since ∼30% of inherited genetic disorders are caused by frameshift or nonsense mutations that generate premature termination codons (PTCs). Therefore, effectors of NMD are potential targets for therapeutic intervention (Frischmeyer and Dietz 1999;Holbrook et al. 2004).Genetic screens in the nematode Caenorhabditis elegans identified seven genes that are required for the degradation of nonsense mutant mRNAs of the unc-54 myosin heavy chain gene (Hodgkin et al. 1989;Pulak and Anderson 1993;Cali et al. 1999). In addition to their suppression-of-unc phenotype, these mutations cause abnormal morphogenesis of the male bursa and the hermaphrodite vulva, and accordingly these genes were termed smg-1-7 (for suppressor with morphological effect on genitalia). In the yeast Sac...

No abstract

The Caenorhabditis elegans cuticle collagens are encoded by a multigene family of between 50 and 100 members and are the major component of the nematode cuticular exoskeleton. They are synthesized in the hypodermis prior to secretion and incorporation into the cuticle and exhibit complex patterns of spatial and temporal expression. We have investigated the cis regulatory requirements for tissue-and stage-specific expression of the cuticle collagen gene dpy-7 and have identified a compact regulatory element which is sufficient to specify hypodermal cell reporter gene expression. This element appears to be a true tissue-specific promoter element, since it encompasses the dpy-7 transcription initiation sites and functions in an orientationdependent manner. We have also shown, by interspecies transformation experiments, that the dpy-7 cis regulatory elements are functionally conserved between C. elegans and C. briggsae, and comparative sequence analysis supports the importance of the regulatory sequence that we have identified by reporter gene analysis. All of our data suggest that the spatial expression of the dpy-7 cuticle collagen gene is established essentially by a small tissue-specific promoter element and does not require upstream activator or repressor elements. In addition, we have found the DPY-7 polypeptide is very highly conserved between the two species and that the C. briggsae polypeptide can function appropriately within the C. elegans cuticle. This finding suggests a remarkably high level of conservation of individual cuticle components, and their interactions, between these two nematode species.Caenorhabditis elegans produces a series of five distinct cuticles, the first of which is secreted by the underlying hypodermis during late embryogenesis and is subsequently shed and replaced at each of the four larval molts. The nematode hypodermis is a complex tissue comprising the external epithelial cells of the organism, and many of these cells are multinucleate syncytia arising from cell fusions which occur during development. The largest of these is hyp-7, which forms the outer surface of the main body region. In the newly hatched L1 larva, hyp-7 contains 23 nuclei, which increase in number throughout development to 133 in the adult as a result of fusions with the progeny of lateral (seam cells [H1, H2, and V cells]) and ventral (P cells) hypodermal blast cells (38,