We have identified and cloned the Caenorhabditis elegans dpy-2 and dpy-10 genes and determined that they encode collagens. Genetic data suggested that these genes are important in morphogenesis and possibly other developmental events. These data include the morphologic phenotypes exhibited by mutants, unusual genetic interactions with the sqt-1 collagen gene, and suppression of mutations in the glp-1 and mup-i genes. The proximity of the dpy-2 and dpy-10 genes (3.5 kilobase) and the structural similarity of their encoded proteins (41% amino acid identity) indicate that dpy-2 and dpy-10 are the result of a gene duplication event. The genes do not, however, appear to be functionally redundant, because a dpy-10 null mutant is not rescued by the dpy-2 gene. In addition, full complementation between dpy-2 and dpy-10 can be demonstrated with all recessive alleles tested in trans. Sequence analysis of several mutant alleles of each gene was performed to determine the nature of the molecular defects that can cause the morphologic phenotypes. Glycine substitutions within the Gly-X-Y portion of the collagens can result in dumpy (Dpy), dumpy, left roller (DLRol), or temperature-sensitive DLRol phenotypes. dpy-1O(cn64), a dominant temperature-sensitive DLRol allele, creates an Arg-to-Cys substitution in the amino non-Gly-X-Y portion of the protein. Three dpy-10 alleles contain Tcl insertions in the coding region of the gene. dpy-lO(cg36) (DLRol) creates a nonsense codon near the end of the Gly-X-Y region. The nature of this mutation, combined with genetic data, indicates that DLRol is the null phenotype of dpy-10. The Dpy phenotype results from reduced function of the dpy-1O collagen gene. Our results indicate that a variety of molecular defects in these collagens can result in severe morphologic changes in C. elegans.

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis ekgans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites.Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.The dinucleotide AG is an essential feature of the 3' splice acceptor site of eukaryotic pre-mRNA introns. A comparison of approximately 3,700 pre-mRNA introns from vertebrates, invertebrates, plants, yeasts, and viruses shows that the AG at the 3' end of introns is virtually invariant: all contain the ultimate nucleotide G, and only four exceptions (involving alternative splice sites) to the penultimate nucleotide A were found (27, 50). All the introns of the nematode Caenorhabditis elegans analyzed to date also end with the dinucleotide AG (15).Consistent with its striking conservation, the invariant AG is functionally critical for proper splicing. Alteration of the invariant AG to either AA, AU, CG, GG, or UG inhibits splicing in vitro (1,35,45). The importance of the dinucleotide AG is also supported by in vivo studies. Introns harboring point mutations in the AG dinucleotide are abnormally spliced in yeasts (1), Neurospora crassa (34), and mammalian cells (25,38). Conversely, mutations that create an AG (e.g., AC mutated to AG) sometimes result in the selection of the newly created AG as a new 3' splice site (36, 57).The necessity of having an AG at the 3' end of introns also has implications for numerous human disorders. Diseases associated with point mutation of either the A or the G at the 3' end of an intron include analbuminemia (47), ,-thalassemia (4), citrullinemia (30, 52), cystic fibrosis (21), debrisoquine polymorphism (22), hemophilia (9), familial type III hyperlipoproteinemia (10), lipoprotein lipase deficiency (23), ornithine transcarbamylase deficiency (8), osteogenesis imperfecta (58), Tay-Sachs disease (39), and xeroderma pigmentosum (49).The in vivo consequences of altering the AG at a 3' splice * Corresponding author. ...

No abstract

No abstract

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.