Genetic Evidence for Interaction between Cbp1 and Specific Nucleotides in the 5′ Untranslated Region of Mitochondrial Cytochrome <i>b</i> mRNA in <i>Saccharomyces cerevisiae</i>

Chen, Wei; Dieckmann, Carol L.

doi:10.1128/mcb.17.11.6203

Cited by 22 publications

(31 citation statements)

References 42 publications

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“…As expected, the ACG and ACG-ACG strains did not grow on glycerol at 30°C after 4 days, although spontaneous revertants arose in both samples as observed previously for the ACG strain (15).…”

Section: Tandem Cbp1supporting

confidence: 53%

“…Our data strongly suggest that Pet127 stimulates trimming of the COB 5Ј-UTR in a 5Ј 3 3Ј-directed exonuclease reaction until it is obstructed by Cbp1. When the interaction between Cbp1 and COB mRNA is abolished by replacing the CCG trinucleotide element with ACG, COB mRNA is degraded from the 5Ј-end (15). Absence of Pet127 leads to the almost complete disappearance of mature COB mRNA and to the enrichment of the precursor form, demonstrating the key role of Pet127 in mitochondrial mRNA maturation.…”

mentioning

confidence: 95%

“…Primer Extension Analysis of mRNA 5Ј-Ends-Primers COB6B and cox4242 were end-labeled with T4 polynucleotide kinase and [␥- 32 P]ATP as recommended by the supplier of the enzyme (Fermentas), and primer extensions were done as previously described (15). Total cellular RNA was isolated from yeast strains after they grew to an optical density at 600 nm of 0.4 to 0.5 (18).…”

Section: Construction Of Plasmids-cob 5ј-utr Sequences Frommentioning

confidence: 99%

“…Genetic studies suggest that Cbp1 binds directly to a sequence within the AU-rich COB mRNA 5Ј-UTR that contains a unique CCG trinucleotide (15). The proximity of the CCG trinucleotide (Ϫ944 to Ϫ942) to the 5Ј-end of the mature COB mRNA prompted the hypothesis that Cbp1 binding protects the mRNA from degradation by blocking a nuclease.…”

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confidence: 99%

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Pet127 Governs a 5′ → 3′-Exonuclease Important in Maturation of Apocytochrome b mRNA in Saccharomyces cerevisiae

Fekete

Ellis

Schonauer

et al. 2008

Journal of Biological Chemistry

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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...

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Section: Tandem Cbp1supporting

confidence: 53%

mentioning

confidence: 95%

Section: Construction Of Plasmids-cob 5ј-utr Sequences Frommentioning

confidence: 99%

mentioning

confidence: 99%

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Pet127 Governs a 5′ → 3′-Exonuclease Important in Maturation of Apocytochrome b mRNA in Saccharomyces cerevisiae

Fekete

Ellis

Schonauer

et al. 2008

Journal of Biological Chemistry

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“…We have hypothesized that Cbp1 interacts with COB RNAs in the region containing the CCG trinucleotide. ACG and CCU mutant strains are temperaturesensitive for respiration and have very low levels of COB mRNA, whereas the CAG mutant strain is respiratory-incompetent at all temperatures and has undetectable levels of mRNA (8). ⌬pet127 null mutations arose as spontaneous suppressors of the conditionally respiratory-deficient ACG and CCU mutations but were unable to suppress the CAG mutation.…”

mentioning

confidence: 99%

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

Islas-Osuna¹,

Ellis²,

Marnell³

et al. 2002

Journal of Biological Chemistry

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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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RNA processing and degradation mechanisms shaping the mitochondrial transcriptome of budding yeasts

Golik

2023

IUBMB Life

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Yeast mitochondrial genes are expressed as polycistronic transcription units that contain RNAs from different classes and show great evolutionary variability. The promoters are simple, and transcriptional control is rudimentary. Posttranscriptional mechanisms involving RNA maturation, stability, and degradation are thus the main force shaping the transcriptome and determining the expression levels of individual genes. Primary transcripts are fragmented by tRNA excision by RNase P and tRNase Z, additional processing events occur at the dodecamer site at the 3′ end of protein‐coding sequences. groups I and II introns are excised in a self‐splicing reaction that is supported by protein splicing factors encoded by the nuclear genes, or by the introns themselves. The 3′‐to‐5′ exoribonucleolytic complex called mtEXO is the main RNA degradation activity involved in RNA turnover and processing, supported by an auxiliary 5′‐to‐3′ exoribonuclease Pet127p. tRNAs and, to a lesser extent, rRNAs undergo several different base modifications. This complex gene expression system relies on the coordinated action of mitochondrial and nuclear genes and undergoes rapid evolution, contributing to speciation events. Moving beyond the classical model yeast Saccharomyces cerevisiae to other budding yeasts should provide important insights into the coevolution of both genomes that constitute the eukaryotic genetic system.

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Genetic Evidence for Interaction between Cbp1 and Specific Nucleotides in the 5′ Untranslated Region of Mitochondrial Cytochrome b mRNA in Saccharomyces cerevisiae

Cited by 22 publications

References 42 publications

Pet127 Governs a 5′ → 3′-Exonuclease Important in Maturation of Apocytochrome b mRNA in Saccharomyces cerevisiae

Pet127 Governs a 5′ → 3′-Exonuclease Important in Maturation of Apocytochrome b mRNA in Saccharomyces cerevisiae

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

RNA processing and degradation mechanisms shaping the mitochondrial transcriptome of budding yeasts

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