A C-terminal fragment of an intron-encoded maturase is sufficient for promoting group I intron splicing

Group I introns often encode proteins that catalyze site-specific DNA hydrolysis. Some of these proteins have acquired the ability to promote splicing of their cognate intron, but whether these two activities reside in different regions of the protein remains obscure. A crystal structure of I-AniI, a dual function intron-encoded protein, has shown that the protein has two pseudo-symmetric domains of equal size. Each domain contacts its DNA substrate on either side of two cleavage sites. As a first step to identify the RNA binding surface, the N- and C-terminal domains of I-AniI were separately expressed and tested for promoting the splicing of the mitochondrial (mt) COB pre-RNA. The N-terminal protein showed no splicing activation or RNA binding, suggesting that this domain plays a minimal role in activity or is improperly folded. Remarkably, the 16-kDa C-terminal half facilitates intron splicing with a rate similar to that of the full-length protein. Both the C-terminal fragment and full-length proteins bind tightly to the COB intron. RNase footprinting shows that the C-terminal and full-length proteins bind to the same regions and induce the same conformational changes in the COB intron. Together, these results show that the C-terminal fragment of I-AniI is necessary and sufficient for maturase activity and suggests that I-AniI acquired splicing function by utilizing a relatively small protein surface that likely represents a novel RNA binding motif. This fragment of I-AniI represents the smallest group I intron splicing cofactor described to date.     

Pentamidine inhibits mitochondrial intron splicing and translation in Saccharomyces cerevisiae

Pentamidine inhibits in vitro splicing of nuclear group I introns from rRNA genes of some pathogenic fungi and is known to inhibit mitochondrial function in yeast. Here we report that pentamidine inhibits the self-splicing of three group I and two group II introns of yeast mitochondria. Comparison of yeast strains with different configurations of mitochondrial introns (12, 5, 4, or 0 introns) revealed that strains with the most introns were the most sensitive to growth inhibition by pentamidine on glycerol medium. Analysis of blots of RNA from yeast strains grown in raffinose medium in the presence or absence of pentamidine revealed that the splicing of seven group I and two group II introns that have intron reading frames was inhibited by the drug to varying extents. Three introns without reading frames were unaffected by the drug in vivo, and two of these were inhibited in vitro, implying that the drug affects splicing by acting directly on RNA in vitro, but on another target in vivo. Because the most sensitive introns in vivo are the ones whose splicing depends on a maturase encoded by the intron reading frames, we tested pentamidine for effects on mitochondrial translation. We found that the drug inhibits mitochondrial but not cytoplasmic translation in cells at concentrations that inhibit mitochondrial intron splicing. Therefore, pentamidine is a potent and specific inhibitor of mitochondrial translation, and this effect explains most or all of its effects on respiratory growth and on in vivo splicing of mitochondrial introns.     

A novel mechanism for protein-assisted group I intron splicing

Previously it was shown that the Aspergillus nidulans (A.n.) mitochondrial COB intron maturase, I-AniI, facilitates splicing of the COB intron in vitro. In this study, we apply kinetic analysis of binding and splicing along with RNA deletion analysis to gain insight into the mechanism of I-AniI facilitated splicing. Our results are consistent with I-AniI and A.n. COB pre-RNA forming a specific but labile encounter complex that is resolved into the native, splicing-competent complex. Significantly, kinetic analysis of splicing shows that the resolution step is rate limiting for splicing. RNA deletion studies show that I-AniI requires most of the A.n. COB intron for binding suggesting that the integrity of the I-AniI-binding site depends on overall RNA tertiary structure. These results, taken together with the observation that A.n. COB intron lacks significant stable tertiary structure in the absence of protein, support a model in which I-AniI preassociates with an unfolded COB intron via a "labile" interaction that facilitates correct folding of the intron catalytic core, perhaps by resolving misfolded RNAs or narrowing the number of conformations sampled by the intron during its search for native structure. The active intron conformation is then "locked in" by specific binding of I-Anil to its intron interaction site.     

The identification of apocytochrome b as a mitochondrial gene product and immunological evidence for altered apocytochrome b in yeast strains having mutations in the COB region of mitochondrial DNA.

The yeast mitochondrial translation product of Mr 30 000 is identical with apocytochrome b. After labelling in vivo with [35S]sulphate in the presence of cycloheximide, the radioactivity in this product present in solubilized submitochondrial particles, was completely recovered in pure cytochrome bc1 complex as a single polypeptide. We show that this translation product is identical with apocytochrome b using peptide mapping by limited proteolysis according to Cleveland et al. [J. Biol. Chem. 250 (1977) 8236-8242] and by immunoprecipitation with a specific antiserum against apocytochrome b. New mitochondrial translation products in 36 strains of Saccharomyces cerevisiae having mutations in the COB region of the mitochondrial DNA, are precipitated by this antiserum. This is consistent with the assumption that many of the cob mutations are localized in the structural gene for apolcytochrome b on mitochondrial DNA. Mutations in two intervening sequences can give rise to products related to apocytochrome b that are considerably longer than normal apocytochrome b. We discuss the hypothesis that in these mutants splicing of the messenger RNA does not occur correctly and that, as a consequence of this, ribosomes read through in an intervening sequence.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

Mne1 is a novel component of the mitochondrial splicing apparatus responsible for processing of a COX1 group I intron in yeast

Saccharomyces cerevisiae cells lacking Mne1 are deficient in intron splicing in the gene encoding the Cox1 subunit of cytochrome oxidase but contain wild-type levels of the bc(1) complex. Thus, Mne1 has no role in splicing of COB introns or expression of the COB gene. Northern experiments suggest that splicing of the COX1 aI5β intron is dependent on Mne1 in addition to the previously known Mrs1, Mss116, Pet54, and Suv3 factors. Processing of the aI5β intron is similarly impaired in mne1Δ and mrs1Δ cells and overexpression of Mrs1 partially restores the respiratory function of mne1Δ cells. Mrs1 is known to function in the initial transesterification reaction of splicing. Mne1 is a mitochondrial matrix protein loosely associated with the inner membrane and is found in a high mass ribonucleoprotein complex specifically associated with the COX1 mRNA even within an intronless strain. Mne1 does not appear to have a secondary function in COX1 processing or translation, because disruption of MNE1 in cells containing intronless mtDNA does not lead to a respiratory growth defect. Thus, the primary defect in mne1Δ cells is splicing of the aI5β intron in COX1.     

Function of the C-terminal domain of the DEAD-box protein Mss116p analyzed in vivo and in vitro

The DEAD-box proteins CYT-19 in Neurospora crassa and Mss116p in Saccharomyces cerevisiae are general RNA chaperones that function in splicing mitochondrial group I and group II introns and in translational activation. Both proteins consist of a conserved ATP-dependent RNA helicase core region linked to N and C-terminal domains, the latter with a basic tail similar to many other DEAD-box proteins. In CYT-19, this basic tail was shown to contribute to non-specific RNA binding that helps tether the core helicase region to structured RNA substrates. Here, multiple sequence alignments and secondary structure predictions indicate that CYT-19 and Mss116p belong to distinct subgroups of DEAD-box proteins, whose C-terminal domains have a defining extended α-helical region preceding the basic tail. We find that mutations or C-terminal truncations in the predicted α-helical region of Mss116p strongly inhibit RNA-dependent ATPase activity, leading to loss of function in both translational activation and RNA splicing. These findings suggest that the α-helical region may stabilize and/or regulate the activity of the RNA helicase core. By contrast, a truncation that removes only the basic tail leaves high RNA-dependent ATPase activity and causes only a modest reduction in translation and RNA splicing efficiency in vivo and in vitro. Biochemical analysis shows that deletion of the basic tail leads to weaker non-specific binding of group I and group II intron RNAs, and surprisingly, also impairs RNA-unwinding at saturating protein concentrations and nucleotide-dependent tight binding of single-stranded RNAs by the RNA helicase core. Together, our results indicate that the two sub-regions of Mss116p's C-terminal domain act in different ways to support and modulate activities of the core helicase region, whose RNA-unwinding activity is critical for both the translation and RNA splicing functions.     

The Cbp2 protein stimulates the splicing of the omega intron of yeast mitochondria.

The Cbp2 protein is encoded in the nucleus and is required for the splicing of the terminal intron of the mitochondrial COB gene in Saccharomyces cerevisiae . Using a yeast strain that lacks this intron but contains a related group I intron in the precursor of the large ribosomal RNA, we have determined that Cbp2 protein is also required for the normal accumulation of 21S ribosomal RNA in vivo . Such strains bearing a deletion of the CBP2 gene adapt slowly to growth in glycerol/ethanol media implying a defect in derepression. At physiologic concentrations of magnesium, Cbp2 stimulates the splicing of the ribosomal RNA intron in vitro . Nevertheless, Cbp2 is not essential for splicing of this intron in mitochondria nor is it required in vitro at magnesium concentrations >5 mM. A similar intron exists in the large ribosomal RNA (LSU) gene of Saccharomyces douglasii . This intron does need Cbp2 for catalytic activity in physiologic magnesium. Similarities between the LSU introns and COB intron 5 suggest that Cbp2 may recognize conserved elements of the these two introns, and protein-induced UV crosslinks occur in similar sites in the substrate and catalytic domains of the RNA precursors.     

Saccharomyces cerevisiae translational activator Cbs1p is associated with translationally active mitochondrial ribosomes

In the yeast Saccharomyces cerevisiae, mitochondrial translation of most, if not all, mitochondrially encoded genes is regulated by an individual set of gene-specific activators. Translation of the COB mRNA encoding cytochrome b requires the function of two nuclearly encoded proteins, Cbs1p and Cbs2p. Genetic data revealed that the 5'-untranslated region of COB mRNA is the target of both proteins. Recently, we provided evidence for an interaction of Cbs2p with mitochondrial ribosomes. We demonstrate here by means of blue native gel electrophoresis, density gradient centrifugation and tandem affinity purification that a portion of Cbs1p is also associated with mitochondrial ribosomes. In addition, we demonstrate that the amount of ribosome-associated Cbs1p is elevated in the presence of chloramphenicol, which is known to stall ribosomes on mRNAs. In the presence of puromycin, which strips off the mRNA and nascent protein chains from ribosomes, Cbs1p is no longer associated with ribosomes. Our data indicate that the observed interaction is mediated by ribosome-bound mRNA, thus restricting the association to ribosomes actively translating cytochrome b.