Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5 -untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species. In an aep3-disruptant strain, the shorter ATP8/6 mRNA was undetectable, and the level of the longer mRNA was reduced to ϳ35% that of wild type. Localization of a hemagglutinin-tagged version of Aep3p showed that the protein is an extrinsic constituent of the mitochondrial inner membrane facing the matrix.

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...

No abstract

The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3-dodecamer sequence, but the 5-ends have seven different sequences. To investigate whether apocytochrome b (COB) mRNA is processed at the 5-end from a longer precursor by an endonuclease or an exonuclease, a 64-nucleotide sequence, which is required for the protection of COB mRNA by the Cbp1 protein and is found at the 5-end of the processed COB mRNA, was duplicated in tandem. The wild-type 64-nucleotide element functioned in either the upstream or downstream position when paired with a mutant element. In the tandem wild-type strain, the 5-end of the mRNA was at the 5-end of the upstream unit, demonstrating that the mRNA is processed by an exonuclease. Accumulation of precursor COB RNA in single and double element strains with a deletion of PET127 demonstrated that the encoded protein governs the 5-exonuclease responsible for processing the precursor to the mature form.In Saccharomyces cerevisiae, mitochondrial apocytochrome b (COB) 3 mRNA is transcribed from the tRNA glu -COB operon and is extensively processed before it reaches its mature form. Many aspects of this process have been well described; at least 15 nuclearly encoded proteins have been identified that control the metabolism of COB mRNA. As depicted in Fig. 1, the initial COB precursor is transcribed from position Ϫ1566, with the A of the AUG codon of COB defined as ϩ1, and contains both tRNA glu and COB mRNA (1, 2). Mitochondrial RNase P and a tRNA 3Ј-endonuclease cleave the initial precursor at positions Ϫ1170 and Ϫ1098, respectively, releasing tRNA glu from the precursor mRNA (3, 4). The mature mRNA is generated by further processing of the precursor RNA at position Ϫ955 or Ϫ954 (5). This trimming step and stabilization of the mature mRNA are not completely understood; however, it is known that the nuclearly encoded protein Cbp1 specifically controls the turnover (5-9) and translation (10) of COB mRNA. In cbp1 mutant strains, the level of tRNA glu is close to that of wild type, whereas precursor COB mRNA is reduced to 25% of wild-type levels and mature COB mRNA is undetectable.The best candidate for the mitochondrial nuclease that processes the COB precursor at Ϫ954/Ϫ955 is a mitochondrial membrane-bound protein, Pet127 (11). It is a member of a large protein family that is well conserved in fungi and protists but is not present in plants or mammals (12). In pet127 strains, 5Ј-end processing of COB, VAR1, and ATP8/6 mRNAs, 15 S rRNA (11), and RPM1, the mitochondrial RNase P RNA 4 (13) is blocked and unprocessed precursor RNAs accumulate to levels equivalent to the sum of processed and unprocessed RNAs in wild-type strains. These defects in processing only inhibit respiratory growth when the temperature is raised to 37°C (11). Pet127 could itself be the RNase that trims these 5Ј-ends, but the protein has no RNase signature sequences or domains. Alternatively, Pet127 c...

Previous studies have suggested that surface components of papillary thyroid carcinoma (PTC) cells may be aberrantly glycanated, but the precise nature of these molecules has not been unveiled nor documented to be of clinical relevance. A monoclonal antibody was raised against a unique keratan sulfate (KS) determinant and used to differentially screen benign and malignant thyroid tissue for the expression of components carrying these moieties. In a total of 349 cases of benign and malignant thyroid lesions, 100% of the 115 PTC cases examined (including various histological subtypes) were found to contain KSbearing molecules, whereas these were virtually absent from benign tissues and other thyroid tumors, with the exception of 21% of the follicular carcinoma cases analyzed. A composite immunoaffinity chromatography, immunochemistry, and mass spectrometric approach revealed that the PTC-specific KS-bearing macromolecules were unique glycoforms of thyroglobulin and transferrin. Combined, reciprocal immunoprecipitation and Western blotting further indicated that the former glycoform predominated and that most of the transferrin produced by PTC was glycanated with KS moieties. Fluorescent keratanase II-based fingerprinting of the KS moieties bound to these isoforms further demonstrated several PTC-specific peculiarities: 1) that a considerable portion of the moieties was covalently attached via a novel core protein linkage structure; 2) they had an unusual extended average length; 3) an unusual relative ratio of highly sulfated disaccharides terminating with ␣ Papillary thyroid carcinoma (PTC) is by far the most frequent malignant tumor of the thyroid with a threefold prevalence in females.