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

Abstract

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...

DMR1 (CCM1/YGR150C) of Saccharomyces cerevisiae Encodes an RNA-Binding Protein from the Pentatricopeptide Repeat Family Required for the Maintenance of the Mitochondrial 15S Ribosomal RNA

“…In principle, disruption of COB and COX1 mRNA maturation would lead to respiratory deficiency, but the stability of the mitochondrial genome should not be affected. There are several examples of nuclear-encoded factors involved in the splicing of COB and/or COX1 introns, such as Mss18p (Seraphin et al 1988), Mrs1p (Bousquet et al 1990), or Cbp2p (Gampel et al 1989), and while they are required for respiratory competence in strains containing their target introns, their dysfunctions do not lead to the loss of mtDNA integrity. An interesting exception is the NAM2 gene, which encodes the mitochondrial leucyl-tRNA synthetase, which is also a splicing factor for the excision of several group I introns (Labouesse et al 1985;Labouesse 1990), but in this case, it is the function in mitochondrial translation that is essential for the integrity of the mitochondrial DNA.…”

Group I Ribozymes: Substrate Recognition, Catalytic Strategies, and Comparative Mechanistic Analysis

“…It self-splices at high MgClz concentration in vitro, whereas splicing under physiological conditions requires CBP2. a protein known to be required for splicing in vivo (McGraw and Tzagoloff 1983;Gampel and Tzagoloff 1987;Partono and Lewin 1988;Gampel et al 1989). Kinetic analysis of the protein-free and protein-facilitated reactions has revealed that splicing is accelerated three orders of magnitude by saturating CBP2 protein at SmMMgCl, (Weeks and Cech 1995a).…”

COX24 Codes for a Mitochondrial Protein Required for Processing of the COX1 Transcript

“…Excision of some intervening sequences also depends on nuclear genes such as CBP2, and SUV3 and MSS116, the latter two coding for RNA helicases (3,21,22). Cbp2p interacts with and stabilizes a splicing competent secondary structure of the cytochrome b pre-mRNA (23). Suv3p has been implicated in stabilizing the COX1 transcript by regulating turnover of group I intronic RNAs (24,25), whereas Mss116p has been shown to function in splicing of all mtDNA introns in S. cerevisiae and Neurospora crassa (26).…”