Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae

Kreike, Jan; Schulze, Marion; Pillar, Timothy M.; Körte, Andreas; Rödel, Gerhard

doi:10.1007/bf00420605

Cited by 47 publications

(22 citation statements)

References 36 publications

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“…The second gene is MRS1 (25,26), whose protein product is encoded in the nucleus, targeted to the mitochondria, and homologous with the nuclear-encoded mitochondrial DNAresolving enzyme, Cce1 (27,28). Cce1 recognizes, manipulates, and cleaves four-way DNA junctions (27) and is a member of the RNase H fold family of DNA junction-resolving enzymes (29,30).…”

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confidence: 99%

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

Bassi

Oliveira

White

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Detectable splicing by the Saccharomyces cerevisiae mitochondrial bI3 group I intron RNA in vitro is shown to require both an intron-encoded protein, the bI3 maturase, and the nuclearencoded protein, Mrs1. Both proteins bind independently to the bI3 RNA. The bI3 maturase binds as a monomer, whereas Mrs1 is a dimer in solution that assembles as two dimers, cooperatively, on the RNA. The active six-subunit complex has a molecular mass of 420 kDa, splices with a k cat of 0.3 min ؊1 , and binds the guanosine nucleophile with an affinity comparable to other group I introns. The functional bI3 maturase domain is translated from within the RNA that encodes the intron, has evolved a high-affinity RNAbinding activity, and is a member of the LAGLIDADG family of DNA endonucleases, but appears to have lost DNA cleavage activity. Mrs1 is a divergent member of the RNase H fold superfamily of dimeric DNA junction-resolving enzymes that also appears to have lost its nuclease activity and now functions as a tetramer in RNA binding. Thus, the bI3 ribonucleoprotein is the product of a process in which a once-catalytically active RNA now obligatorily requires two facilitating protein cofactors, both of which are compromised in their original functions. M any small ribozymes, both those that occur naturally (1) and those that are products of in vitro selection experiments (2), display an impressive array of catalytic activities. Although these simple structures are capable of performing catalysis, most cellular ribozymes have undergone a process of elaboration in which potentially simple RNA active sites are bolstered by additional RNA structural domains (3, 4) or protein cofactors (5-9).One example of this process is the group I introns. The group I intron RNA core consists of two extended and roughly coaxially stacked helices that form a catalytic cleft, which binds, in turn, the 5Ј and 3Ј splice site helices and the guanosine cofactor (guanosine 5Ј-monophosphate, pG) (4, 5, 10). Although the catalytic core is relatively compact and can independently form a functional active site (11), most group I introns have acquired additional peripheral structures that function to stabilize the RNA core (4, 12, 13). Moreover, whereas a large number of group I introns have been identified by sequence and structural analysis (4, 14), both anecdotal and experimental evidence suggests that many have lost their self-splicing activity (15). It appears that group I intron RNAs commonly recruit protein cofactors and now function as obligatory ribonucleoproteins (8, 16).The Saccharomyces cerevisiae mitochondrial cytochrome b bI3 group I intron represents an especially intriguing case of elaboration by accretion of peripheral RNA domains and protein cofactors. First, the bI3 RNA potentially spans two group I intron subgroups, IA2 and IB4, characterized by RNA sequence insertions between helices P7 and P3 and adjacent to P9 and by an extension of the P5 helix, respectively (4) (Fig. 1A, boxed structures). The bI3 intron also contains two tertiary inter...

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Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

Bassi

Oliveira

White

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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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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“…Despite the ability of some group I introns to self-splice in vitro (4), genetic evidence indicates that in many cases proteins are required for splicing in vivo (9,12,27,29). One such documented case is exemplified by the terminal intron of the yeast mitochondrial cytochrome b pre-mRNA which is capable of self-splicing in vitro (14, 37) but requires a protein encoded by the yeast nuclear gene CBP2 for in vivo splicing (23,33).…”

Section: Discussionmentioning

confidence: 99%

“…Although some mitochondrial group I introns are able to self-splice (splice in the absence of protein) in vitro, there is compelling evidence that in vivo splicing requires the participation of protein factors. In Saccharomyces cerevisiae, at least 11 nuclear gene products have been reported to be required for splicing of mitochondrial transcripts (27,33,40). In addition, some group I introns in yeast mitochondrial genes contain open reading frames that code for proteins (maturases) involved in the excision of the cognate intervening sequences (IVSs) from the pre-mRNAs (2, 12, 29).…”

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confidence: 99%

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

Gampel¹,

Nishikimi²,

Tzagoloff³

1989

Mol. Cell. Biol.

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The terminal intron (b12) of the yeast mitochondrial cytochrome b gene is a group I intron capable of self-splicing in vitro at high concentrations of Mg2+. Excision of b12 in vivo, however, requires a protein encoded by the nuclear gene CBP2. The CBP2 protein has been partially purified from wild-type yeast mitochondria and shown to promote splicing at physiological concentrations of Mg2+. The self-splicing and protein-dependent splicing reactions utilized a guanosine nucleoside cofactor, the hallmark of group I intron self-splicing reactions. Furthermore, mutations that abolished the autocatalytic activity of b12 also blocked protein-dependent splicing. These results indicated that protein-dependent excision of b12 is an RNA-catalyzed process involving the same two-step transesterification mechanism responsible for self-splicing of group I introns. We propose that the CBP2 protein binds to the bI2 precursor, thereby stabilizing the catalytically active structure of the RNA. The same or a similar RNA structure is probably induced by high concentrations of Mg2e in the absence of protein. Binding of the CBP2 protein to the unspliced precursor was supported by the observation that the protein-dependent reaction was saturable by the wild-type precursor. Proteindependent splicing was competitively inhibited by excised bI2 and by a splicing-defective precursor with a mutation in the 5' exon near the splice site but not by a splicing-defective precursor with a mutation in the core structure. Binding of the CBP2 protein to the precursor RNA had an effect on the 5' splice site helix, as evidenced by suppression of the interaction of an exogenous dinucleotide with the internal guide sequence.A substantial number of genes in fungal mitochondrial DNA contain group I introns (35). Although some mitochondrial group I introns are able to self-splice (splice in the absence of protein) in vitro, there is compelling evidence that in vivo splicing requires the participation of protein factors. In Saccharomyces cerevisiae, at least 11 nuclear gene products have been reported to be required for splicing of mitochondrial transcripts (27,33,40). In addition, some group I introns in yeast mitochondrial genes contain open reading frames that code for proteins (maturases) involved in the excision of the cognate intervening sequences (IVSs) from the pre-mRNAs (2, 12, 29). Protein-facilitated splicing is not confined to yeast mitochondrial transcripts. Excision of several mitochondrial group I introns of Neurospora crassa depends on a protein encoded by the nuclear gene cytl8 (9). This protein has been partially purified and shown to promote splicing of the large rRNA precursor in vitro (1, 16).The terminal intron (bI2) of the yeast mitochondrial cytochrome b pre-mRNA is an example of a group I intron whose in vivo excision depends on a nuclear gene product (bI2 of the short cytochrome b gene is identical to b15 of the long gene). Mutations in a gene designated CBP2 have been shown to selectively block processing of b12 (23, 33). In vitro...

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Cloning of a nuclear gene MRS1 involved in the excision of a single group I intron (bI3) from the mitochondrial COB transcript in S. cerevisiae

Cited by 47 publications

References 36 publications

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

Recruitment of intron-encoded and co-opted proteins in splicing of the bI3 group I intron RNA

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

CBP2 protein promotes in vitro excision of a yeast mitochondrial group I intron.

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