N-Ethyl-N-Nitrosourea (ENU) Mutagenesis Reveals an Intronic Residue Critical for Caenorhabditis elegans 3′ Splice Site Function in Vivo
| Autor: | Chunfang Guo, Thomas Blumenthal, Omar A. Itani, Stephane Flibotte, Patrick J. Hu, Kathleen J. Dumas |
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| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
RNA Splicing Factors polypyrimidine tract ENU Investigations QH426-470 Polymorphism Single Nucleotide 03 medical and health sciences splicing Genetics Animals splice Histone octamer Nucleotide Motifs Caenorhabditis elegans Molecular Biology Genetics (clinical) Sequence Deletion Splice site mutation biology Base Sequence 3′ splice site Intron Chromosome Mapping biology.organism_classification Introns daf-12 030104 developmental biology Phenotype Polypyrimidine tract Mutagenesis Ethylnitrosourea RNA splicing Mutation C. elegans RNA Splice Sites Protein Binding |
| Zdroj: | G3: Genes, Genomes, Genetics, Vol 6, Iss 6, Pp 1751-1756 (2016) G3: Genes|Genomes|Genetics |
| ISSN: | 2160-1836 |
| DOI: | 10.1534/g3.116.028662 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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