Two group I mitochondrial introns in the cob-box and coxI genes require the same MRS1/PET157 nuclear gene product for splicing

Bousquet, Isabelle; Dujardin, Geneviève; Poyton, Robert O.; Słonimski, Piotr P.

doi:10.1007/bf00312599

Cited by 48 publications

(30 citation statements)

References 33 publications

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“…Mutations were cloned in a yeast expression plasmid in-frame with the Myc tag at the C-terminal end and transformed into the MRS1 deletion strain. In addition to aI5␤ processing, Mrs1p also functions in splicing of the bI3 group I intron, but otherwise is dispensable for respiratory function (23,38,39). As such, growth defects on nonfermentable carbon are directly due to impaired splicing (see below).…”

Section: Resultsmentioning

confidence: 99%

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Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

Turk

Caprara

2010

Journal of Biological Chemistry

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Yeast Suv3p is a member of the DEXH/D box family of RNA helicases and is a critical component of the mitochondrial degradosome, which also includes a 3 3 5 exonuclease, Dss1p. Defects in the degradosome result in accumulation of aberrant transcripts, unprocessed transcripts, and excised group I introns. In addition, defects in SUV3 result in decreased splicing of the aI5␤ and bI3 group I introns. Whereas a role for Suv3p in RNA degradation is well established, the function of Suv3p in splicing of group I introns has remained elusive. It has been particularly challenging to determine if Suv3p effects group I intron splicing through RNA degradation as part of the degradosome, or has a direct role in splicing as a chaperone, because nearly all perturbations of SUV3 or DSS1 result in loss of the mitochondrial genome. Here we utilized the suv3-1 allele, which is defective in RNA metabolism and yet maintains a stable mitochondrial genome, to investigate the role of Suv3p in splicing of the aI5␤ group I intron. We provide genetic evidence that Mrs1p is a limiting cofactor for aI5␤ splicing, and this evidence also suggests that Suv3p activity is required to recycle the excised aI5␤ ribonucleoprotein. We also show that Suv3p acts indirectly as a component of the degradosome to promote aI5␤ splicing. We present a model whereby defects in Suv3p result in accumulation of stable, excised group I intron ribonucleoproteins, which result in sequestration of Mrs1p, and a concomitant reduction in splicing of aI5␤.Mitochondrial gene expression is largely controlled by posttranscriptional mechanisms because of the relatively simple nature of mitochondrial transcription (1-5). One critical control point is RNA degradation, which is involved in the extensive processing required of mitochondrial transcripts (6, 7). In Saccharomyces cerevisiae, intergenic regions are removed from polycistronic transcripts and in the case of two mRNAs, encoding the cytochrome oxidase I subunit (COX1) 2 and apocytochrome b subunit (COB), group I and group II catalytic introns are also removed. Splicing of these introns requires trans-acting factors to facilitate the RNA-catalyzed intron excision and ligation of flanking exons (8).Rapid turnover of excised group I introns is essential for mitochondrial gene expression because these RNAs act as endonucleases that cleave their parent, processed RNAs at the site of exon ligation (e.g. exon reopening reactions), which decreases expression (9). Degradation of excised group I introns is carried out by a two-protein complex called the mitochondrial degradosome or mtEXO (10, 11). The mitochondrial degradosome includes Dss1p, a 3Ј 3 5Ј single-stranded exonuclease, and Suv3p, a DEXH/D protein, one of the largest family of proteins that are involved in RNA metabolism (11-13). Indeed, some members show ATP-dependent unwinding of RNA helices. Suv3p is a member of the SKI2 subfamily, individuals of which are thought to couple ATP binding and hydrolysis cycles with translocation along an RNA single strand and disruption...

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Section: Resultsmentioning

confidence: 99%

“…However, like SUV3, disruption of the MRS1 gene results in splicing defects for both introns (23,39). Here we show a strong genetic interaction between MRS1 and SUV3 in that overexpression of MRS1 partially suppressed splicing defects in the suv3-1 strain.…”

Section: Discussionmentioning

confidence: 99%

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

Turk

Caprara

2010

Journal of Biological Chemistry

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“…Expression of COB requires participation of nuclearly encoded proteins. For example, Cbp2 (28,42), Mrs1 (5,21,22), Mrs2 (20,43), and Nam2 (24,26) are required for intron splicing; Cbp1 stabilizes COB mRNA (8,12); and Cbs1 (29,36) and Cbs2 (Cbp7) (29,34,35) are COB-specific translation factors. None of these nuclearly encoded factors is essential for growth of yeast on fermentable carbon sources (e.g., glucose), but strains with null mutations in the genes encoding these factors have a PET (petite) phenotype; they form smaller colonies than the wild-type strains on glucose media.…”

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confidence: 99%

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

Chen

Dieckmann

1997

Molecular and Cellular Biology

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The cytochrome b (COB) gene is encoded by the mitochondrial genome; however, its expression requires the participation of several nuclearly encoded protein factors. The yeast Cbp1 protein, which is encoded by the nuclear CBP1 gene, is required for the stabilization of COB mRNA. A previous deletion analysis identified an 11-nucleotide-long sequence within the 5 untranslated region of COB mRNA that is important for Cbp1-dependent COB mRNA stability. In the present study, site-directed mutagenesis experiments were carried out to define further the features of this cis element. The CCG sequence within this region was shown to be necessary for stability. A change in residue 533 of Cbp1 from aspartate to tyrosine suppresses the effects of a single-base change in the CCG element. This is strong genetic evidence that the nuclearly encoded Cbp1 protein recognizes and binds directly to the sequence containing CCG and thus protects COB mRNA from degradation.Mitochondrial biogenesis is a coordinated process that requires the function of both nuclear and mitochondrial gene products. For example, cytochrome b is an electron carrier located in the inner membrane of mitochondria along with other components of the respiratory chain and is encoded by the mitochondrial gene COB. Expression of COB requires participation of nuclearly encoded proteins. For example, Cbp2 (28, 42), Mrs1 (5,21,22), Mrs2 (20,43), and Nam2 (24, 26) are required for intron splicing; Cbp1 stabilizes COB mRNA (8, 12); and Cbs1 (29, 36) and Cbs2 (Cbp7) (29,34,35) are COB-specific translation factors. None of these nuclearly encoded factors is essential for growth of yeast on fermentable carbon sources (e.g., glucose), but strains with null mutations in the genes encoding these factors have a PET (petite) phenotype; they form smaller colonies than the wild-type strains on glucose media. PET mutants cannot grow on nonfermentable carbon sources (e.g., glycerol or ethanol), which require mitochondrial function for utilization.In yeast mitochondria, the genes for tRNA Glu and cytochrome b are cotranscribed as a unit (Fig. 1). Transcription begins at position Ϫ1566, with the A of the AUG initiation codon of COB defined as ϩ1. RNase P and tRNA 3Ј endonuclease process the initial transcript at the 5Ј and 3Ј ends of tRNA Glu , respectively (7, 18), which releases tRNA Glu and COB precursor RNA with a 5Ј end extending to Ϫ1098. Further processing of COB precursor RNA generates the mature COB message with a 5Ј end at Ϫ955 or Ϫ954 (4).Cbp1 was identified because it is required for the stabilization of COB mRNA (12). In cbp1 mutant strains, the level of tRNA Glu is wild type, COB precursor RNA is decreased to ϳ20% of the wild-type level, and the mature COB message is undetectable. Since inadequate apocytochrome b is produced, cbp1 mutant strains cannot respire. We previously used deletion analysis to locate a small region in the COB 5Ј UTR (untranslated region) which is essential for two Cbp1-dependent functions: positioning of the Ϫ955/Ϫ954 cleavage site and COB message ...

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“…Cce1 is a structure-specific DNA endonuclease, while Mrs1 is thought to have a nonenzymatic function in stabilizing the tertiary conformation of a self-splicing group I intron (13). Presumably, one was recruited to perform the role of the other.…”

Section: Discussionmentioning

confidence: 99%

“…cerevisiae Cce1 has previously been observed to share 29% sequence identity with the yeast protein Mrs1 (5). Mrs1 (Pet157) is a nuclear encoded protein from S. cerevisiae that is required for splicing of two group I introns in the mitochondrion (12,13). It may function in stabilizing the folded structure of the group I intron to promote self-splicing, as has been shown for the protein Cbp2 (14,15).…”

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confidence: 99%

Site-directed Mutagenesis of the Yeast Resolving Enzyme Cce1 Reveals Catalytic Residues and Relationship with the Intron-splicing Factor Mrs1

Wardleworth

Kvaratskhelia

White

2000

Journal of Biological Chemistry

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The Holliday junction-resolving enzyme Cce1 is a magnesium-dependent endonuclease, responsible for the resolution of recombining mitochondrial DNA molecules in Saccharomyces cerevisiae. We have identified a homologue of Cce1 from Candida albicans and used a multiple sequence alignment to predict residues important for junction binding and catalysis. Twelve site-directed mutants have been constructed, expressed, purified, and characterized. Using this approach, we have identified basic residues with putative roles in both DNA recognition and catalysis of strand scission and acidic residues that have a purely catalytic role. We have shown directly by isothermal titration calorimetry that a group of acidic residues vital for catalytic activity in Cce1 act as ligands for the catalytic magnesium ions. Sequence similarities between the Cce1 proteins and the group I intron splicing factor Mrs1 suggest the latter may also possess a binding site for magnesium, with a putative role in stabilization of RNA tertiary structure or catalysis of the splicing reaction.

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Two group I mitochondrial introns in the cob-box and coxI genes require the same MRS1/PET157 nuclear gene product for splicing

Cited by 48 publications

References 33 publications

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

Splicing of Yeast aI5β Group I Intron Requires SUV3 to Recycle MRS1 via Mitochondrial Degradosome-promoted Decay of Excised Intron Ribonucleoprotein (RNP)

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

Site-directed Mutagenesis of the Yeast Resolving Enzyme Cce1 Reveals Catalytic Residues and Relationship with the Intron-splicing Factor Mrs1

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