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RNA processing and expression of an intron-encoded protein in yeast mitochondria: role of a conserved dodecamer
Abstract:
The 3' ends of most Saccharomyces cerevisiae mitochondrial mRNAs terminate at a conserved dodecamer sequence, 5'-AAUAAUAUUCUU-3', of unknown function. We have studied the consequences of mutations within a dodecamer found in an 1,143-base-pair optional intron of the mitochondrial large (21S) rRNA gene on RNA processing. The dodecamer is situated at the 3' end of an expressed open reading frame (ORF) within that intron, and the mutations are two adjacent transversions that extend the intron ORF by 51 nucleotides. The strain harboring these mutations, L5-10-1, is defective in biased intron transmission in crosses to strains that lack the intron, as are other mutants which contain nucleotide changes within the ORF (I. G. Macreadie, R. M. Scott, A. R. Zinn, and R. A. Butow, Cell 41:395-402, 1985). However, unlike these other mutants, wild-type strains, or petites which retain the intron allele, L5-10-1 is defective in processing at the intron dodecamer. In addition, L5-10-1 lacks a prominent 2.7-kilobase RNA containing both intron and exon sequences and at least two of four RNAs that correspond to various forms of the excised intron. We propose that these RNAs, missing in L5-10-1 but present in all other strains examined, arise in part by processing at the intron dodecamer. In addition, in all strains examined, we have detected a novel processing activity in which precursor 21S rRNA transcripts are cleaved in the upstream exon, about 1,500 nucleotides from the 5' end of the RNA. This activity, together with 3' intron dodecamer cleavage, probably accounts for the 2.7-kilobase RNA species, a candidate for the mRNA for the intron-encoded protein.
Insights
Mutations in a conserved dodecamer sequence within Saccharomyces cerevisiae mitochondrial introns disrupt RNA processing and the production of specific RNA species. This study investigates the function of this dodecamer in intron RNA processing and transmission.
Area of Science:
- Molecular Biology
- Yeast Genetics
- RNA Processing
Background:
- Most Saccharomyces cerevisiae mitochondrial mRNAs end at a conserved dodecamer sequence (5'-AAUAAUAUUCUU-3') of unknown function.
- An optional intron in the mitochondrial large (21S) rRNA gene contains this dodecamer at the 3' end of an expressed open reading frame (ORF).
Purpose of the Study:
- To investigate the function of the intron dodecamer sequence in Saccharomyces cerevisiae mitochondrial RNA processing.
- To analyze the consequences of mutations within the intron dodecamer on RNA processing and intron transmission.
Main Methods:
- Site-directed mutagenesis of the intron dodecamer sequence in the Saccharomyces cerevisiae mitochondrial 21S rRNA gene.
- Analysis of RNA processing intermediates and products using Northern blotting.
- Assessment of intron transmission efficiency in yeast crosses.
Main Results:
- Mutations in the intron dodecamer (strain L5-10-1) resulted in defective processing at the dodecamer site.
- L5-10-1 mutant strains lacked a specific 2.7-kilobase RNA and several forms of the excised intron RNA.
- A novel processing activity cleaving precursor 21S rRNA in the upstream exon was detected in all strains.
Conclusions:
- The intron dodecamer plays a crucial role in the processing of intron-derived RNAs in Saccharomyces cerevisiae mitochondria.
- Processing at the intron dodecamer, along with upstream cleavage, likely generates a 2.7-kilobase RNA, a potential mRNA for the intron-encoded protein.
- The study elucidates a novel RNA processing pathway in yeast mitochondria involving both dodecamer cleavage and upstream exon cleavage.