title: N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.
abstract: Metazoan introns contain a polypyrimidine tract immediately upstream of the AG dinucleotide that defines the 3' splice site. In the nematode Caenorhabditis elegans, 3' splice sites are characterized by a highly conserved UUUUCAG/R octamer motif. While the conservation of pyrimidines in this motif is strongly suggestive of their importance in pre-mRNA splicing, in vivo evidence in support of this is lacking. In an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in Caenorhabditis elegans, we have isolated a strain containing a point mutation in the octamer motif of a 3' splice site in the daf-12 gene. This mutation, a single base T-to-G transversion at the -5 position relative to the splice site, causes a strong daf-12 loss-of-function phenotype by abrogating splicing. The resulting transcript is predicted to encode a truncated DAF-12 protein generated by translation into the retained intron, which contains an in-frame stop codon. Other than the perfectly conserved AG dinucleotide at the site of splicing, G at the -5 position of the octamer motif is the most uncommon base in C. elegans 3' splice sites, occurring at closely paired sites where the better match to the splicing consensus is a few bases downstream. Our results highlight both the biological importance of the highly conserved -5 uridine residue in the C. elegans 3' splice site octamer motif as well as the utility of using ENU as a mutagen to study the function of polypyrimidine tracts and other AU- or AT-rich motifs in vivo.
authors.affiliation: Institute of Gerontology and Departments of Internal Medicine and Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Omar A
authors.initials: OA
authors.lastname: Itani
authors.affiliation: Department of Zoology, University of British Columbia, Vancouver, British Columbia V6T 123, Canada.
authors.forename: Stephane
authors.initials: S
authors.lastname: Flibotte
authors.affiliation: Institute of Gerontology and Departments of Internal Medicine and Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Kathleen J
authors.initials: KJ
authors.lastname: Dumas
authors.affiliation: Institute of Gerontology and Departments of Internal Medicine and Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109.
authors.forename: Chunfang
authors.initials: C
authors.lastname: Guo
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309.
authors.forename: Thomas
authors.initials: T
authors.lastname: Blumenthal
authors.affiliation: Institute of Gerontology and Departments of Internal Medicine and Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109 pathu@umich.edu.
authors.forename: Patrick J
authors.identifier: 0000-0002-5611-8062
authors.initials: PJ
authors.lastname: Hu
mesh_terms: D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D002874:Chromosome Mapping; D005038:Ethylnitrosourea / Q000633:toxicity*; D007438:Introns; D016296:Mutagenesis / Q000187:drug effects*; D009154:Mutation; D059372:Nucleotide Motifs; D010641:Phenotype; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D022821:RNA Splice Sites; D000072260:RNA Splicing Factors / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D022821:RNA Splice Sites; D000072260:RNA Splicing Factors; D005038:Ethylnitrosourea
keywords: 3′ splice site; C. elegans; ENU; daf-12; polypyrimidine tract; splicing