The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation

1989 ◽  
Vol 9 (10) ◽  
pp. 4161-4169
Author(s):  
S A Mayer ◽  
C L Dieckmann
Keyword(s):  
Carbon Source ◽  
Cytochrome B ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Chain Complex ◽  
Complex Iii ◽  
Yeast Cells ◽  
Transcriptional Level ◽  
Amino Acid Residues ◽  
Gene Encoding

CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA. Cytochrome b is the only mitochondrially synthesized component of the respiratory chain complex III. Since the nuclearly encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated. To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly encoded complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a nonfermentable carbon source. Poly(A)+ RNA from derepressed yeast cells was examined by Northern (RNA) analyses with cRNA probes from CBP1. Both 2.2- and 1.3-kilobase (kb) transcripts were detected. The 1.3-kb mRNA lacked approximately 900 nucleotides of the 3' end of the 2.2-kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence. Northern analyses of RNA isolated from deletion-insertion mutants of CBP1 and from strains that overexpress CBP1 mRNA demonstrated that both mRNAs were transcribed from the CBP1 gene. Furthermore, we demonstrated that the levels of the two CBP1 mRNAs were reciprocally regulated by the carbon source in the growth medium. This is the first description of a yeast gene from which two transcripts that can encode proteins with distinctly different coding properties are generated by alternative 3'-end formation.

scholarly journals The yeast CBP1 gene produces two differentially regulated transcripts by alternative 3'-end formation.

1989 ◽  
Vol 9 (10) ◽  
pp. 4161-4169 ◽  
Author(s):  
S A Mayer ◽  
C L Dieckmann
Keyword(s):  
Carbon Source ◽  
Cytochrome B ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Chain Complex ◽  
Complex Iii ◽  
Yeast Cells ◽  
Transcriptional Level ◽  
Amino Acid Residues ◽  
Gene Encoding

CBP1 is a yeast nuclear gene encoding a mitochondrial protein that stabilizes the 5' end of cytochrome b (cob) pre-mRNA. Cytochrome b is the only mitochondrially synthesized component of the respiratory chain complex III. Since the nuclearly encoded subunits of this complex are regulated at the transcriptional level by catabolite repression, we hypothesized that CBP1 might be similarly regulated. To test the idea that transcriptional regulation of CBP1 could coordinate an increase in cytochrome b mRNA stability with an increase in nuclearly encoded complex III subunit production, we characterized the change in abundance of CBP1 mRNA during derepression on a nonfermentable carbon source. Poly(A)+ RNA from derepressed yeast cells was examined by Northern (RNA) analyses with cRNA probes from CBP1. Both 2.2- and 1.3-kilobase (kb) transcripts were detected. The 1.3-kb mRNA lacked approximately 900 nucleotides of the 3' end of the 2.2-kb mRNA, which encodes the carboxyl-terminal 250 amino acid residues of the CBP1 coding sequence. Northern analyses of RNA isolated from deletion-insertion mutants of CBP1 and from strains that overexpress CBP1 mRNA demonstrated that both mRNAs were transcribed from the CBP1 gene. Furthermore, we demonstrated that the levels of the two CBP1 mRNAs were reciprocally regulated by the carbon source in the growth medium. This is the first description of a yeast gene from which two transcripts that can encode proteins with distinctly different coding properties are generated by alternative 3'-end formation.

Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function

1991 ◽  
Vol 11 (2) ◽  
pp. 813-821
Author(s):  
S A Mayer ◽  
C L Dieckmann
Keyword(s):  
Mitochondrial Function ◽  
Developmental Stages ◽  
Single Gene ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
State Level ◽  
Yeast Cells ◽  
Cdna Clones ◽  
Coding Sequence ◽  
Polyadenylation Sites

Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.

Saccharomyces cerevisiae contains two functional citrate synthase genes

1986 ◽  
Vol 6 (6) ◽  
pp. 1936-1942
Author(s):  
K S Kim ◽  
M S Rosenkrantz ◽  
L Guarente
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Carbon Source ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Nuclear Gene ◽  
Tricarboxylic Acid ◽  
Yeast Genome ◽  
Gene Encoding ◽  
Acid Cycle ◽  
Nonfermentable Carbon Source

The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed.

The NAM9-1 suppressor mutation in a nuclear gene encoding ribosomal mitochondrial protein of Saccharomyces cerevisiae

Gene ◽  
1995 ◽  
Vol 162 (1) ◽  
pp. 81-85 ◽  
Author(s):  
Aleksandra Dmochowska ◽  
Agata Konopińska ◽  
Magdalena Krzymowska ◽  
Barbara Szcześniak ◽  
Magdalena Boguta
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Suppressor Mutation ◽  
Gene Encoding

scholarly journals Role of mitochondria in the pheromone- and amiodarone-induced programmed death of yeast

2005 ◽  
Vol 168 (2) ◽  
pp. 257-269 ◽  
Author(s):  
Andrei I. Pozniakovsky ◽  
Dmitry A. Knorre ◽  
Olga V. Markova ◽  
Anthony A. Hyman ◽  
Vladimir P. Skulachev ◽  
...  
Keyword(s):  
Mitochondrial Protein ◽  
Chain Complex ◽  
Energy Coupling ◽  
Complex Iii ◽  
Strong Increase ◽  
Cytochrome C Release ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Doses ◽  
Multicellular Organisms

Although programmed cell death (PCD) is extensively studied in multicellular organisms, in recent years it has been shown that a unicellular organism, yeast Saccharomyces cerevisiae, also possesses death program(s). In particular, we have found that a high doses of yeast pheromone is a natural stimulus inducing PCD. Here, we show that the death cascades triggered by pheromone and by a drug amiodarone are very similar. We focused on the role of mitochondria during the pheromone/amiodarone-induced PCD. For the first time, a functional chain of the mitochondria-related events required for a particular case of yeast PCD has been revealed: an enhancement of mitochondrial respiration and of its energy coupling, a strong increase of mitochondrial membrane potential, both events triggered by the rise of cytoplasmic [Ca2+], a burst in generation of reactive oxygen species in center o of the respiratory chain complex III, mitochondrial thread-grain transition, and cytochrome c release from mitochondria. A novel mitochondrial protein required for thread-grain transition is identified.

scholarly journals Yeast CBP1 mRNA 3' end formation is regulated during the induction of mitochondrial function.

1991 ◽  
Vol 11 (2) ◽  
pp. 813-821 ◽  
Author(s):  
S A Mayer ◽  
C L Dieckmann
Keyword(s):  
Mitochondrial Function ◽  
Developmental Stages ◽  
Single Gene ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
State Level ◽  
Yeast Cells ◽  
Cdna Clones ◽  
Coding Sequence ◽  
Polyadenylation Sites

Alternative mRNA processing is one mechanism for generating two or more polypeptides from a single gene. While many mammalian genes contain multiple mRNA 3' cleavage and polyadenylation signals that change the coding sequence of the mature mRNA when used at different developmental stages or in different tissues, only one yeast gene has been identified with this capacity. The Saccharomyces cerevisiae nuclear gene CPB1 encodes a mitochondrial protein that is required for cytochrome b mRNA stability. This 66-kDa protein is encoded by a 2.2-kb mRNA transcribed from CPB1. Previously we showed that a second 1.2-kb transcript is initiated at the CBP1 promoter but has a 3' end near the middle of the coding sequence. Furthermore, it was shown that the ratio of the steady-state level of 2.2-kb CBP1 message to 1.2-kb message decreases 10-fold during the induction of mitochondrial function, while the combined levels of both messages remain constant. Having proposed that regulation of 3' end formation dictates the amount of each CBP1 transcript, we now show that a 146-bp fragment from the middle of CBP1 is sufficient to direct carbon source-regulated production of two transcripts when inserted into the yeast URA3 gene. This fragment contains seven polyadenylation sites for the wild-type 1.2-kb mRNA, as mapped by sequence analysis of CBP1 cDNA clones. Deletion mutations upstream of the polyadenylation sites abolished formation of the 1.2-kb transcript, whereas deletion of three of the sites only led to a reduction in abundance of the 1.2-kb mRNA. Our results indicate that regulation of the abundance of both CBP1 transcripts is controlled by elements in a short segment of the gene that directs 3' end formation of the 1.2-kb transcript, a unique case in yeast cells.

scholarly journals Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit of the mitochondrial ribosome.

1988 ◽  
Vol 8 (9) ◽  
pp. 3647-3660 ◽  
Author(s):  
J A Partaledis ◽  
T L Mason
Keyword(s):  
Catabolite Repression ◽  
Genomic Library ◽  
Gene Dosage ◽  
Function Analysis ◽  
Mitochondrial Protein ◽  
Nuclear Gene ◽  
Small Subunit ◽  
Transcriptional Level ◽  
Coding Region ◽  
Mitochondrial Ribosome

MRP13 is defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in lambda gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment. Sequencing of this fragment showed that the MRP13 coding region specifies a 324-amino-acid basic protein with a calculated Mr of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired in either mitochondrial protein synthesis or assembly of 37S ribosomal subunits, indicating that, like L29 and L30 in Escherichia coli (M. Lotti, E. R. Dabbs, R. Hasenbank, M. Stöffler-Meilicke, and G. Stöffler, Mol. Gen. Genet. 192:295-300, 1983), MRP13 is not essential for ribosome synthesis or function. Analysis of the sequence in the MRP13 5'-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein rp39A. The rp39A coding region began at nucleotide -846 and ended at -325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In [rho+] cells, transcript levels were repressed severalfold by growth in glucose compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains ([rho0], [mit-]), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Despite high levels of the MRP13 transcripts in [rho0] cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein in a significant but lesser amount. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration and, in addition, that protein levels appear to be modulated posttranscriptionally by degradation of free copies of the MRP13 protein.

scholarly journals Identification of a Novel Variant in EARS2 Associated with a Severe Clinical Phenotype Expands the Clinical Spectrum of LTBL

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1028
Author(s):  
Sofia Barbosa-Gouveia ◽  
Emiliano González-Vioque ◽  
Álvaro Hermida ◽  
María Unceta Suarez ◽  
María Jesús Martínez-González ◽  
...  
Keyword(s):  
Clinical Presentation ◽  
Protein Biosynthesis ◽  
Mitochondrial Protein ◽  
Mitochondrial Disorder ◽  
Nuclear Gene ◽  
Chain Complex ◽  
Trna Synthetase ◽  
Clinical Spectrum ◽  
Genes Encoding ◽  
Novel Variant

The EARS2 nuclear gene encodes mitochondrial glutamyl-tRNA synthetase, a member of the class I family of aminoacyl-tRNA synthetases (aaRSs) that plays a crucial role in mitochondrial protein biosynthesis by catalyzing the charging of glutamate to mitochondrial tRNA(Glu). Pathogenic EARS2 variants have been associated with a rare mitochondrial disorder known as leukoencephalopathy with thalamus and brainstem involvement and high lactate (LTBL). The targeted sequencing of 150 nuclear genes encoding respiratory chain complex subunits and proteins implicated in the oxidative phosphorylation (OXPHOS) function was performed. The oxygen consumption rate (OCR), and the extracellular acidification rate (ECAR), were measured. The enzymatic activities of Complexes I-V were analyzed spectrophotometrically. We describe a patient carrying two heterozygous EARS2 variants, c.376C>T (p.Gln126*) and c.670G>A (p.Gly224Ser), with infantile-onset disease and a severe clinical presentation. We demonstrate a clear defect in mitochondrial function in the patient’s fibroblasts, suggesting the molecular mechanism underlying the pathogenicity of these EARS2 variants. Experimental validation using patient-derived fibroblasts allowed an accurate characterization of the disease-causing variants, and by comparing our patient’s clinical presentation with that of previously reported cases, new clinical and radiological features of LTBL were identified, expanding the clinical spectrum of this disease.

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