RRC ID 53269
Author Itani OA, Flibotte S, Dumas KJ, Guo C, Blumenthal T, Hu PJ.
Title N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3' Splice Site Function in Vivo.
Journal G3 (Bethesda)
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.
Volume 6(6)
Pages 1751-6
Published 2016-6-1
DOI 10.1534/g3.116.028662
PII g3.116.028662
PMID 27172199
PMC PMC4889670
MeSH Animals Base Sequence Caenorhabditis elegans / drug effects* Caenorhabditis elegans / genetics* Caenorhabditis elegans / metabolism Chromosome Mapping Ethylnitrosourea / toxicity* Introns* Mutagenesis / drug effects* Mutation Nucleotide Motifs Phenotype Polymorphism, Single Nucleotide Protein Binding RNA Splice Sites* RNA Splicing Factors / metabolism Sequence Deletion
IF 2.63
Times Cited 1
C.elegans tm3188