Generation of Temperature - Sensitive cbpl Strains of Saccharomyces cerevisiae by PCR Mutagenesis and in Vivo Recombination : Characteristics of the Mutant Strains Imply That CBPl Is Involved in Stabilization and Processing of Cytochrome b Pre - mRNA Robin

Mitochondrial biogenesis is dependent on both nuclearly and mitochondrially encoded proteins. Study of the nuclearly encoded mitochondrial gene products and their effect on mitochondrial genome expression is essential to understanding mitochondrial function. Mutations in the nuclear gene CBPl of Saccharomyces cerevisiae result in degradation of mitochondrially encoded cytochrome b (cob) RNA; thus, the cells are unable to respire. Putative roles for the CBPl protein include processing of precursor RNA to yield the mature 5' end of cob mRNA and/or physical protection of the mRNA from degradation by nucleases. To examine the activity of CBPl , we generated temperature-sensitive cbpl mutant strains by polymerase chain reaction (PCR) mutagenesis and i vivo recombination. These temperature-sensitive cbpl strains lack cob mRNA only at the nonpermissive temperature. Quantitative primer extension analyses of RNA from these strains and from a cbpl deletion strain demonstrated that CBPl is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBPl is not involved solely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are reduced only slightly at the nonpermissive temperature. Collectively, these data lead us to favor a hypothesis whereby CBPl protects cob precursor RNAs and promotes the processing event that generates the mature 5' end of the mRNA. I . M ITOCHONDRIAL biogenesis is dependent upon both nuclearly encoded and mitochondrially encoded proteins (for review see COSTANZO and Fox 1990; GRIVELL 1989; TZAGOLOFF and MYERS 1986). In the yeast Saccharomyces cerevisiae the expression of mitochondrial genes is a complex process involving hundreds of nuclear P E T genes (TZAGOLOFF and DIECKMANN 1990). Some P E T gene products participate in transcription and translation and are not specific to particular mitochondrial genes, whereas others affect the expression of individual mitochondrial genes. These gene-specific proteins may coordinate the expression of the two genomes to yield a functional mitochondrion. To understand mitochondrial-nuclear interactions, we must understand gene-specific interactions. A number of nuclearly encoded factors are required specifically for expression of the mitochondrial gene encoding cytochrome b (cob) of S. cerevisiae. The nuclearly encoded mitochondrial proteins CBS l , CBS2 and CBP6 are required for translation of cob mRNA (DIECKMANN and TZAGOLOFF 1985; RODEL 1986; RODEL and FOX 1987). Although cob mRNA is present in these strains, the messages are not translated. Strains with mutations in the nuclear gene C B P l are respiratory deficient because cob transcripts are deGenetics 135: 981-991 (December, 1993) graded (DIECKMANN, HOMISON and TZAGOLOFF 1984; DIECKMANN, KOERNER and TZAGOLOFF 1984; DIECKMANN and MITTELMEIER 1987; DIECKMANN, PAPE and TZAGOLOFF 1982; MITTELMEIER and DIECKMANN 1990). Therefore, CBPl is required for the accumulation of stable cob mRNA. Supportive of a direct role in cob transcript maturation or stability, CBPl has been shown to be imported into yeast mitochondria (WEBER and DIECKMANN 1990). It has been hypothesized that CBPl either protects cob mRNA from degradation, or processes precursor RNA to generate stable cob mRNA (DIECKMANN, HOMISON and TZAGOLOFF 1984; DIECKMANN, KOERNER and TZAGOLOFF 1984). Mature cob mRNA is processed from a primary transcript as shown in Figure 1 (BONITZ et al. 1982; CHRISTIANSON et al. 1983). Transcription of the tRNAg'"-cob unit and processing of tRNAg'" are not affected in cbpl strains (DIECKMANN, KOERNER and TZAGOLOFF 1984). Following removal of tRNAg'" (CHEN and MARTIN 1988; HOLLINGSWORTH and MARTIN 1986), the resulting transcript with a 5' end at -1098 (relative to +I of the initiating AUG) is processed further at -954 to generate the mature 5' end of cob mRNA. Recently, sequence between -961 and -898 of the untranslated leader of cob has been shown to be sufficient for CBPl982 R. R. Staples and C. L. Dieckmann