Disruption of the yeast nuclear PET54 gene blocks excision of mitochondrial intron aI5 beta from pre-mRNA for cytochrome c oxidase subunit I.
The nuclear PET54 gene of Saccharomyces cerevisiae was cloned and a pet54::LEU2 gene disruption strain was constructed. Analysis of the phenotype of this strain revealed a defect in expression of two mitochondrial genes: COX1, which encodes cytochrome c oxidase subunit I, and COX3, which encodes cytochrome c oxidase subunit III. The defect in COX1 gene expression in the pet54 mutant was shown to be the result of inefficient excision of COX1 intron aI5 beta. Two lines of evidence indicate that inefficient excision of intron aI5 beta is the sole defect in COX1 gene expression. First, a pet54::LEU2 cytoductant bearing the 'short' mitochondrial genome that lacks both COX1 introns aI5 alpha and aI5 beta is defective only in COX3 gene expression and not in COX1 mRNA splicing or mRNA translation. Second, Northern analysis of COX1 transcipts from the pet54 mutant showed that a 3.8 kb COX1 transcript containing unexcised intron aI5 beta and lacking intron aI5 alpha is accumulated while the amount of 2.2 kb mature COX1 mRNA is diminished. In an effort to relate the role of the PET54 gene product in splicing of COX1 pre-mRNA to the previously characterized role for PET54 in translation of mitochondrial COX3 mRNA, the sequence of the PET54-responsive portion of the COX3 5' untranslated leader region was compared to the COX1 intron aI5 beta sequence. Two blocks of RNA sequence present in COX3 have similar counterparts within intron aI5 beta of COX1. The possibility that the PET54 protein binds to one or the other of these blocks of RNA sequence and the potential consequences of this interaction are discussed.
Valencik
M L
ML
Department of Microbiology, University of California, Los Angeles 90024.
Kloeckener-Gruissem
B
B
Poyton
R O
RO
McEwen
J E
JE
D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004272:DNA, Mitochondrial / Q000235:genetics*; D003576:Electron Transport Complex IV / Q000235:genetics*; D015870:Gene Expression; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D012322:RNA Precursors / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004272:DNA, Mitochondrial; D012322:RNA Precursors; D012333:RNA, Messenger; D003576:Electron Transport Complex IV