Cbp1 Is Required for Translation of the Mitochondrial Cytochromeb mRNA of Saccharomyces cerevisiae

Abstract

Expression of the yeast mitochondrial cytochrome b gene (COB) is controlled by at least 15 nuclear-encoded proteins. One of these proteins, Cbp1, is required for COB mRNA stability. ⌬cbp1 null strains fail to accumulate mature COB mRNA and cannot respire. Since ⌬cbp1 null strains lack mature COB transcripts, the hypothesis that Cbp1 also plays a role in translation of these mRNAs could not be tested previously. 5-End trimming of precursor COB RNA and other mitochondrial transcripts is dependent on Pet127. pet127 mutants accumulate high levels of precursor COB mRNA and have no mature mRNA. pet127 mutants respire well; this phenotype implies that COB precursor RNA is translated efficiently. With the expectation that a ⌬cbp1⌬pet127 strain might accumulate substantial levels of COB RNA, the double null strain was constructed and analyzed to test the hypothesis that Cbp1 is required for translation of COB RNA. The ⌬cbp1⌬pet127 strain does accumulate levels of COB precursor mRNA that are ϳ60% of the level of COB mRNA in the wild-type strain. However, cytochrome b protein is not synthesized, and thus the ⌬cbp1⌬pet127 strain does not respire. These results suggest that Cbp1 is required for translation of COB RNAs.The expression of mitochondrial genes at the level of transcription, RNA processing, translation, post-translational modification, and complex assembly depends on many nuclearencoded proteins that are synthesized in the cytoplasm and imported into mitochondria (1-3). Mutations in these nuclear genes often lead to respiratory deficiency, termed the pet 1 phenotype because colonies are petite in size on fermentable glucose medium. To understand how mitochondrial gene expression is regulated, the function of these nuclear PET genes must first be understood.The nuclear PET gene CBP1 encodes a protein that is imported into mitochondria and is required for the stability of the mitochondrial cytochrome b (COB) mRNA (4, 5). COB mRNA is co-transcribed with the upstream tRNA glu . The tRNA is processed from the initial transcript by mitochondrial RNaseP and tRNA 3Ј-endonuclease, leaving a transcript we have called the COB precursor RNA. The precursor is further shortened at the 5Ј-end to produce what we have called the mature COB mRNA. In a wild-type strain, there is approximately five times more mature than precursor COB RNA. In a cbp1 null strain, the mature COB mRNA is undetectable, and precursor RNA is reduced 2-to 5-fold from wild-type levels ( Fig. 1) (6). cbp1 null strains are respiratory-deficient, and no apocytochrome b is synthesized, which suggests that: the 5Ј-extension on the precursor RNA inhibits translation, the abundance of the precursor is below the threshold required for respiration (about 4% of the levels of mature mRNA in the wild-type strain), or Cbp1 is required for translation of COB RNAs. Evidence that precursor COB RNAs can be translated has come from studies of Pet127, a nuclear-encoded protein that is localized to mitochondria, where it plays a role in RNA processing. A ⌬pet127 null strain exh...

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