GENERATION OF TEMPERATURE-SENSITIVE CBP1 STRAINS OF SACCHAROMYCES-CEREVISIAE BY PCR MUTAGENESIS AND IN-VIVO RECOMBINATION - CHARACTERISTICSOF THE MUTANT STRAINS IMPLY THAT CBP1 IS INVOLVED IN STABILIZATION AND PROCESSING OF CYTOCHROME-B PRE-MESSENGER-RNA

Mitochondrial biogenesis is dependent on both nuclearly and mitochondr ially encoded proteins. Study of the nuclearly encoded mitochondrial g ene products and their effect on mitochondrial genome expression is es sential to understanding mitochondrial function. Mutations in the nucl ear gene CBP1 of Saccharomyces cerevisiae result in degradation of mit ochondrially encoded cytochrome b (cob) RNA; thus, the cells are unabl e to respire. Putative roles for the CBP1 protein include processing o f 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 CBP1 we generated temperature-sensitive cbp1 mutant strain s by polymerase chain reaction (PCR) mutagenesis and in vivo recombina tion. These temperature-sensitive cbp1 strains lack cob mRNA only at t he nonpermissive temperature. Quantitative primer extension analyses o f RNA from these strains and from a cbp1 deletion strain demonstrated that CBP1 is required for the stability of precursor RNAs in addition to production of the stable mature mRNA. Thus, CBP1 is not involved so lely in the protection of mature cob mRNA from nucleases. Moreover, we found that mature mRNAs are undetectable while precursor RNAs are red uced only slightly at the nonpermissive temperature. Collectively, the se data lead us to favor a hypothesis whereby CBP1 protects cob precur sor RNAs and promotes the processing event that generates the mature 5 ' end of the mRNA.