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

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.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4161-4169.1989 ◽

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.

Download Full-text

The human mitochondrial 12S rRNA m4C methyltransferase METTL15 is required for mitochondrial function

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012127 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8505-8513 ◽

Cited By ~ 5

Author(s):

Hao Chen ◽

Zhennan Shi ◽

Jiaojiao Guo ◽

Kao-jung Chang ◽

Qianqian Chen ◽

...

Keyword(s):

Mitochondrial Function ◽

Mitochondrial Protein ◽

Nuclear Gene ◽

Small Subunit ◽

12S Rrna ◽

Mitochondrial Rna ◽

Small Subunit Rrna ◽

Mitochondrial Ribosomes

Mitochondrial DNA gene expression is coordinately regulated both pre- and post-transcriptionally, and its perturbation can lead to human pathologies. Mitochondrial rRNAs (mt-rRNAs) undergo a series of nucleotide modifications after release from polycistronic mitochondrial RNA precursors, which is essential for mitochondrial ribosomal biogenesis. Cytosine N4-methylation (m4C) at position 839 (m4C839) of the 12S small subunit mt-rRNA was identified decades ago; however, its biogenesis and function have not been elucidated in detail. Here, using several approaches, including immunofluorescence, RNA immunoprecipitation and methylation assays, and bisulfite mapping, we demonstrate that human methyltransferase-like 15 (METTL15), encoded by a nuclear gene, is responsible for 12S mt-rRNA methylation at m4C839 both in vivo and in vitro. We tracked the evolutionary history of RNA m4C methyltransferases and identified a difference in substrate preference between METTL15 and its bacterial ortholog rsmH. Additionally, unlike the very modest impact of a loss of m4C methylation in bacterial small subunit rRNA on the ribosome, we found that METTL15 depletion results in impaired translation of mitochondrial protein-coding mRNAs and decreases mitochondrial respiration capacity. Our findings reveal that human METTL15 is required for mitochondrial function, delineate the evolution of methyltransferase substrate specificities and modification patterns in rRNA, and highlight a differential impact of m4C methylation on prokaryotic ribosomes and eukaryotic mitochondrial ribosomes.

Download Full-text

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

Molecular and Cellular Biology ◽

10.1128/mcb.9.10.4161 ◽

1989 ◽

Vol 9 (10) ◽

pp. 4161-4169 ◽

Cited By ~ 18

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.

Download Full-text

Mutations in nuclear gene cyt-4 of Neurospora crassa result in pleiotropic defects in processing and splicing of mitochondrial RNAs.

Genetics ◽

10.1093/genetics/123.1.97 ◽

1989 ◽

Vol 123 (1) ◽

pp. 97-108 ◽

Cited By ~ 1

Author(s):

K F Dobinson ◽

M Henderson ◽

R L Kelley ◽

R A Collins ◽

A M Lambowitz

Keyword(s):

Neurospora Crassa ◽

Mitochondrial Protein ◽

Nuclear Gene ◽

Northern Hybridization ◽

Rrna Gene ◽

Mitochondrial Rna ◽

Group I Introns ◽

Group I ◽

Processing Pathways ◽

Aberrant Rna

Abstract The nuclear cyt-4 mutants of Neurospora crassa have been shown previously to be defective in splicing the group I intron in the mitochondrial large rRNA gene and in 3' end synthesis of the mitochondrial large rRNA. Here, Northern hybridization experiments show that the cyt-4-1 mutant has alterations in a number of mitochondrial RNA processing pathways, including those for cob, coI, coII and ATPase 6 mRNAs, as well as mitochondrial tRNAs. Defects in these pathways include inhibition of 5' and 3' end processing, accumulation of aberrant RNA species, and inhibition of splicing of both group I introns in the cob gene. The various defects in mitochondrial RNA synthesis in the cyt-4-1 mutant cannot be accounted for by deficiency of mitochondrial protein synthesis or energy metabolism, and they suggest that the cyt-4-1 mutant is defective in a component or components required for processing and/or turnover of a number of different mitochondrial RNAs. Defective splicing of the mitochondrial large rRNA intron in the cyt-4-1 mutant may be a secondary effect of failure to synthesize pre-rRNAs having the correct 3' end. However, a similar explanation cannot be invoked to account for defective splicing of the cob pre-mRNA introns, and the cyt-4-1 mutation may directly affect splicing of these introns.

Download Full-text

Cosegregation of Single Genes Associated with Fertility Restoration and Transcript Processing of Sorghum Mitochondrial orf107 and urf209

Genetics ◽

10.1093/genetics/150.1.383 ◽

1998 ◽

Vol 150 (1) ◽

pp. 383-391 ◽

Cited By ~ 1

Author(s):

Hoang V Tang ◽

Ruying Chang ◽

Daryl R Pring

Keyword(s):

Fertility Restoration ◽

Mitochondrial Gene ◽

Pollen Viability ◽

Single Gene ◽

Dominant Gene ◽

Nuclear Gene ◽

Male Sterile ◽

Reading Frame ◽

Transcript Processing ◽

Backcross Populations

Abstract Defective nuclear-cytoplasmic interactions leading to aberrant microgametogenesis in sorghum carrying the IS1112C male-sterile cytoplasm occur very late in pollen maturation. Amelioration of this condition, the restoration of pollen viability, involves a novel two-gene gametophytic system, wherein genes designated Rf3 and Rf4 are required for viability of individual gametes. Rf3 is tightly linked to, or represents, a single gene that regulates a transcript processing activity that cleaves transcriptsof orf107, a chimeric mitochondrial open reading frame specific to IS1112C. The mitochondrial gene urf 209 is also subject to nucleus-specific enhanced transcript processing, 5′ to the gene, conferred by a single dominant gene designated Mmt1. Examinations of transcript patterns in F2 and two backcross populations indicated cosegregation of the augmented orf107 and urf209 processing activities in IS1112C. Several sorghum lines that do not restore fertility or confer orf107 transcript processing do exhibit urf209 transcript processing, indicating that the activities are distinguishable. We conclude that the nuclear gene(s) conferring enhanced orf107 and urf209 processing activities are tightly linked in IS1112C. Alternatively, the similarity in apparent regulatory action of the genes may indicate allelic differences wherein the IS1112C Rf3 allele may differ from alleles of maintainer lines by the capability to regulate both orf107 and urf209 processing activities.

Download Full-text

Cryptic polyadenylation sites within the coding sequence of three yeast genes expressed in tobacco

Gene ◽

10.1016/s0378-1119(99)00544-2 ◽

2000 ◽

Vol 242 (1-2) ◽

pp. 87-95 ◽

Cited By ~ 8

Author(s):

Sébastien Grec ◽

Yingchun Wang ◽

Laurence Le Guen ◽

Valentine Negrouk ◽

Marc Boutry

Keyword(s):

Coding Sequence ◽

Polyadenylation Sites ◽

Yeast Genes

Download Full-text

The Cloning and Mapping of ADR6, a Gene Required for Sporulation and for Expression of the Alcohol Dehydrogenase II Isozyme From Saccharomyces cerevisiae

Genetics ◽

10.1093/genetics/116.4.531 ◽

1987 ◽

Vol 116 (4) ◽

pp. 531-540

Author(s):

Aileen K W Taguchi ◽

Elton T Young

Keyword(s):

Saccharomyces Cerevisiae ◽

Alcohol Dehydrogenase ◽

Single Gene ◽

Carbon Sources ◽

Transcription Unit ◽

Yeast Cells ◽

Recessive Mutation ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Activating Sequence ◽

A Site

ABSTRACT The alcohol dehydrogenase II (ADH2) gene of the yeast, Saccharomyces cerevisiae, is not transcribed during growth on fermentable carbon sources such as glucose. Growth of yeast cells in a medium containing only nonfermentable carbon sources leads to a marked increase or derepression of ADH2 expression. The recessive mutation, adr6-1, leads to an inability to fully derepress ADH2 expression and to an inability to sporulate. The ADR6 gene product appears to act directly or indirectly on ADH2 sequences 3' to or including the presumptive TATAA box. The upstream activating sequence (UAS) located 5' to the TATAA box is not required for the Adr6- phenotype. Here, we describe the isolation of a recombinant plasmid containing the wild-type ADR6 gene. ADR6 codes for a 4.4-kb RNA which is present during growth both on glucose and on nonfermentable carbon sources. Disruption of the ADR6 transcription unit led to viable cells with decreased ADHII activity and an inability to sporulate. This indicates that both phenotypes result from mutations within a single gene and that the adr6-1 allele was representative of mutations at this locus. The ADR6 gene mapped to the left arm of chromosome XVI at a site 18 centimorgans from the centromere.

Download Full-text

The nine amino-terminal residues of delta-aminolevulinate synthase direct beta-galactosidase into the mitochondrial matrix

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.355-364.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 355-364

Author(s):

T Keng ◽

E Alani ◽

L Guarente

Keyword(s):

Amino Acid ◽

Fusion Proteins ◽

Specific Activity ◽

Nuclear Gene ◽

Yeast Cells ◽

Mitochondrial Matrix ◽

Aminolevulinate Synthase ◽

Amino Terminal ◽

Activity Measurements ◽

Beta Galactosidase

delta-Aminolevulinate synthase, the first enzyme in the heme biosynthetic pathway, is encoded by the nuclear gene HEM1. The enzyme is synthesized as a precursor in the cytoplasm and imported into the matrix of the mitochondria, where it is processed to its mature form. Fusions of beta-galactosidase to various lengths of amino-terminal fragments of delta-aminolevulinate synthase were constructed and transformed into yeast cells. The subcellular location of the fusion proteins was determined by organelle fractionation. Fusion proteins were found to be associated with the mitochondria. Protease protection experiments involving the use of intact mitochondria or mitoplasts localized the fusion proteins to the mitochondrial matrix. This observation was confirmed by fractionation of the mitochondrial compartments and specific activity measurements of beta-galactosidase activity. The shortest fusion protein contains nine amino acid residues of delta-aminolevulinate synthase, indicating that nine amino-terminal residues are sufficient to localize beta-galactosidase to the mitochondrial matrix. The amino acid sequence deduced from the DNA sequence of HEM1 showed that the amino-terminal region of delta-aminolevulinate synthase was largely hydrophobic, with a few basic residues interspersed.

Download Full-text

In vivo analysis of sequences necessary for CBP1-dependent accumulation of cytochrome b transcripts in yeast mitochondria

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.4203-4213.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 4203-4213

Author(s):

T M Mittelmeier ◽

C L Dieckmann

Keyword(s):

Cytochrome B ◽

Mitochondrial Gene ◽

Microprojectile Bombardment ◽

Nuclear Gene ◽

State Level ◽

Transcription Unit ◽

Endonucleolytic Cleavage ◽

In Vivo Analysis ◽

Nucleotide Region

In Saccharomyces cerevisiae, cytochrome b, an essential component of the respiratory chain, is encoded by the mitochondrial gene cob. The cob transcription unit includes the tRNA(Glu) gene from positions -1170 to -1099 relative to the cob ATG at +1. The initial tRNA(Glu)-cob transcript undergoes several processing events, including removal of tRNA(Glu) and production of the mature 5' end of cob mRNA at nucleotide -954. The nuclear gene product CBP1 is specifically required for the accumulation of cob mRNA. In cbp1 mutant strains, cob transcripts are not detectable by Northern (RNA) blot analysis, but the steady-state level of tRNA(Glu) is similar to that of wild type. The results of a previous study led to the conclusion that a 400-nucleotide region just downstream of tRNA(Glu) is sufficient for CBP1 function. In the present study, the microprojectile bombardment method of mitochondrial transformation was used to introduce deletions within this region of cob. The analysis of cob transcripts in strains carrying the mitochondrial deletion genomes indicates that a 63-nucleotide sequence that encompasses the cleavage site at -954 is sufficient both for CBP1 function and for correct positioning of the cleavage. Furthermore, the data indicate that CBP1 prevents the degradation of unprocessed cob transcripts produced by endonucleolytic cleavage at the 3' end of tRNA(Glu).

Download Full-text