COB Intron Research Articles

Distribution and Polymorphisms of Group I Introns in Mitochondrial Genes from Cryptococcus neoformans and Cryptococcus gattii.

The species complexes Cryptococcus neoformans and Cryptococcus gattii are the causative agents of cryptococcosis. Virulence and susceptibility to antifungals may vary within each species according to the fungal genotype. Therefore, specific and easily accessible molecular markers are required to distinguish cryptic species and/or genotypes. Group I introns are potential markers for this purpose because they are polymorphic concerning their presence and sequence. Therefore, in this study, we evaluated the presence of group I introns in the mitochondrial genes cob and cox1 in different Cryptococcus isolates. Additionally, the origin, distribution, and evolution of these introns were investigated by phylogenetic analyses, including previously sequenced introns for the mtLSU gene. Approximately 80.5% of the 36 sequenced introns presented homing endonucleases, and phylogenetic analyses revealed that introns occupying the same insertion site form monophyletic clades. This suggests that they likely share a common ancestor that invaded the site prior to species divergence. There was only one case of heterologous invasion, probably through horizontal transfer to C. decagattii (VGIV genotype) from another fungal species. Our results showed that the C. neoformans complex has fewer introns compared to C. gattii. Additionally, there is significant polymorphism in the presence and size of these elements, both among and within genotypes. As a result, it is impossible to differentiate the cryptic species using a single intron. However, it was possible to differentiate among genotypes within each species complex, by combining PCRs of mtLSU and cox1 introns, for C. neoformans species, and mtLSU and cob introns for C. gattii species.

Separate Origins of Group I Introns in Two Mitochondrial Genes of the Katablepharid Leucocryptos marina

Mitochondria are descendants of the endosymbiotic α-proteobacterium most likely engulfed by the ancestral eukaryotic cells, and the proto-mitochondrial genome should have been severely streamlined in terms of both genome size and gene repertoire. In addition, mitochondrial (mt) sequence data indicated that frequent intron gain/loss events contributed to shaping the modern mt genome organizations, resulting in the homologous introns being shared between two distantly related mt genomes. Unfortunately, the bulk of mt sequence data currently available are of phylogenetically restricted lineages, i.e., metazoans, fungi, and land plants, and are insufficient to elucidate the entire picture of intron evolution in mt genomes. In this work, we sequenced a 12 kbp-fragment of the mt genome of the katablepharid Leucocryptos marina. Among nine protein-coding genes included in the mt genome fragment, the genes encoding cytochrome b and cytochrome c oxidase subunit I (cob and cox1) were interrupted by group I introns. We further identified that the cob and cox1 introns host open reading frames for homing endonucleases (HEs) belonging to distantly related superfamilies. Phylogenetic analyses recovered an affinity between the HE in the Leucocryptos cob intron and two green algal HEs, and that between the HE in the Leucocryptos cox1 intron and a fungal HE, suggesting that the Leucocryptos cob and cox1 introns possess distinct evolutionary origins. Although the current intron (and intronic HE) data are insufficient to infer how the homologous introns were distributed to distantly related mt genomes, the results presented here successfully expanded the evolutionary dynamism of group I introns in mt genomes.

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.

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.

Functionally Distinct Nucleic Acid Binding Sites for a Group I Intron Encoded RNA Maturase/DNA Homing Endonuclease

A large number of group I introns encode a family of homologous proteins that either promote intron splicing (maturases) or are site-specific DNA endonucleases that function in intron mobility (a process called “homing”). Genetic studies have shown that some of these proteins have both activities, yet how a single protein carries out both functions remains obscure. The similarity between respective DNA-binding sites and the RNA structure near the 5′ and 3′ splice sites has fueled speculation that such proteins may use analogous interactions to perform both functions. The Aspergillus nidulans mitochondrial COB group I intron encodes a bi-functional protein, I- AniI, that has both RNA maturase and site-specific DNA endonuclease activities in vitro. Here, we show that I- AniI shows distinctive features of the endonuclease family to which it belongs, including highly specific, tight binding and sequential DNA strand cleavage. Competition experiments demonstrate that I- AniI binds the COB intron RNA even in saturating concentrations of its DNA target site substrate, suggesting that the protein has a separate binding site for RNA. In addition, we provide evidence that two different DNA-binding site mutants of I- AniI have little effect on the protein's RNA maturation activity. Since RNA splicing is likely a secondary adaptation of the protein, these observations support a model in which homing endonucleases may have developed maturase function by utilizing a hitherto “non-functional” protein surface.

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 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.

Splicing of COB intron 5 requires pairing between the internal guide sequence and both flanking exons.

Group I introns are characterized by a set of conserved sequence elements and secondary structures. Evidence supporting the pairing of certain of these sequences has come from the comparison of intron sequences and from the analysis of mutations that disrupt splicing by interfering with pairing. One of the structures proposed for all group I introns is an internal guide sequence that base pairs with the upstream and the downstream exons, bringing them into alignment for ligation. We made specific mutations in the internal guide sequence and the flanking exons of the fifth intron in the yeast mitochondrial gene for apocytochrome b (COB). Mutations that disrupted the pairing between the internal guide sequence and the upstream exon (the P1 pairing) blocked addition of guanosine to the 5' end of the intron during autocatalytic reactions and prevented formation of the full-length circular intron. In contrast, transcripts containing mutations that disrupted the pairing between the guide sequence and the downstream exon (the P10 helix) initiated splicing but failed to ligate exons. Compensatory mutations that restored helices of normal stability mitigated the effects of the original mutations. These data provide direct evidence for the importance of the base pairing between the internal guide sequence and the downstream exon in the splicing of a wild-type group I intron.

Distribution of mitochondrial introns in the species Schizosaccharomyces pombe and the origin of the group II intron in the gene encoding apocytochrome b.

The mitochondrial genome size of 26 different Schizosaccharomyces pombe strains varies between 17.6 and 24.6 kilobase pairs due to the presence or absence of introns. One of these is the group II intron in the gene encoding apocytochrome b (cob: intron cobI1). Partial DNA sequences of continuous cob genes from six strains (including strain EF1: Trinkl et al. 1985) revealed identical nucleotide sequence in the region where the group II intron is inserted in the mosaic form of the gene. In contrast, analysis of the mosaic cob gene in strain UCD-FstI revealed several base pair changes in the exon regions flanking the splice point, compared with the continuous genes and with the mosaic cob gene in strain 50 (Lang et al. 1985). The base pair differences between the exons of the two mosaic cob genes and the identity of exons in all continuous cob genes argue in favour of the two cob introns in strains 50 and UCD-FstI as independent later acquisitions of the genes, rather than loss of the intron from a common mosaic ancestor of all strains. Other introns present in some but not all strain include two group I introns without open reading frame in the gene encoding subunit 1 of cytochrome c oxidase (cox1: introns cox1I2a and cox1I3), and two group I introns with open reading frames in the same gene (introns cox1I1 and cox1I2b).

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.

The respiratory deficient yeast nuclear mutant MK3 is defective in the synthesis of the mature transcripts of the mitochondrial COB and OX13 genes, which code for apocytochrome b and subunit I of cytochrome c oxidase, resp. Introns 3 and 4 of the COB transcript (bI3 and bI4) and intron 4 (aI4) of the OXI3 transcript can not be excised (Pillar et al. 1983a, b). When combined with mitochondrial genomes lacking introns bI1, bI2 and bI3, or lacking intron bI3 alone the mutant is respiratory competent. Thus, the non-excision of bI4 and aI4 turns out to be an indirect effect of the mutation. From a wild type yeast genebank a plasmid has been isolated with a 3.3 kb DNA insert, which complements the mutant. Subcloning experiments assigned the functional gene to a 1.6 kb HaeIII-Sau3A fragment. Hybridization experiments showed, that it is (i) a single copy gene, (ii) also present in strain D273-10B, containing the "short form" mitochondrial genome (lacking the COB introns bI1-bI3), and (iii) located on chromosome IX. The nuclear gene defective in mutant MK3, was named MRS1 (Mitochondrial RNA Splicing). The involvement of this nuclear gene in the excision of a single group I mitochondrial intron (bI3) of the COB transcript is discussed.

The mosaic cox1 gene in the mitochondrial genome of Schizosaccharomyces pombe: minimal structural requirements and evolution of group I introns

The gene encoding subunit 1 of cytochrome oxidase ( cox1) in the fission yeast Schizosaccharomyces pombe is polymorphic. In strain 50 it contains two group I introns with open reading frames (ORFs) in phase with the upstream exons (Lang, 1984). In strain EF1 two additional very short group I introns which do not possess ORFs were detected by DNA sequencing. These two introns (AI2a and AI3) share distinct characteristics concerning their nucleotide sequence and secondary structure and are located at identical positions as the introns AI4 and AI5β, respectively, in the cox1 gene of Saccharomyces cerevisiae. The sequence homology of the cob and cox1 genes around the splice points of introns AI2a, AI4, and BI4 ( cob intron 4) might reflect horizontal gene transfer between the distantly related species S. pombe and S. cerevisiae.

RNA splicing in Neurospora mitochondria : Defective splicing of mitochondrial mRNA precursors in the nuclear mutant cyt18-1

cyt18-1 (299-9) is a nuclear mutant of Neurospora crassa that has been shown to have a temperature-sensitive defect in splicing the mitochondrial large rRNA intron. In the present work, we investigate the effect of the cyt18-1 mutation on splicing of mitochondrial mRNA introns. Two genes were studied in detail; the cytochrome b ( cob) gene, which contains two introns, and a “long form” of the cytochrome oxidase subunit I ( coI) gene, which contains four introns. We found that splicing of both cob introns and splicing of at least two of the coI introns are strongly inhibited in the mutant, whereas splicing of col intron 1, which is excised as a 2.6 × 10 3 base circle, is relatively unaffected. The rRNA intron and both cob introns are group I introns, whereas the circular coI intron may belong to another structural class. Control experiments showed that the degree of inhibition of splicing is greater in the mutant than can be accounted for by severe inhibition of mitochondrial protein synthesis. Finally, experiments in which mutant cells were shifted from 25 °C to 37 °C showed that splicing of the large rRNA precursor and splicing of the coI mRNA precursor are inhibited with similar kinetics. Considered together, our results suggest that the cyt18-1 gene encodes a trans-acting component that is required for the splicing of group I mitochondrial DNA introns or some subclass thereof. Since Neurospora cob intron 1 has been shown to be self-splicing in vitro, defective splicing of this intron in cyt18-1 indicates that an essentially RNA-catalyzed splicing reaction must be facilitated by a trans-acting factor, presumably a protein, in vivo.

RNA splicing in neurospora mitochondria: Self-splicing of a mitochondrial intron in vitro

We have used Neurospora nuclear mutant cyt-18-1, which accumulates a number of unspliced mitochondrial precursor RNAs, to identify rapidly mitochondrial introns that are self-splicing in vitro. Incubation of deproteinized whole mitochondrial RNA from the mutant with 32P-GTP resulted in strong labeling of a 1.3 kb RNA, subsequently identified as cytochrome b (cob) intron 1, and weaker labeling of additional RNAs. Self-splicing of cob intron 1, including precise cleavage and ligation, was confirmed using an in vitro transcript synthesized from the SP6 promoter. The in vitro splicing reaction was shown to be analogous to that for the Tetrahymena nuclear rRNA intron. Since splicing of cob intron 1 is inhibited in a recessive nuclear mutant, we infer that this essentially RNA-catalyzed splicing reaction must be facilitated by a protein in vivo.

Processing of yeast mitochondrial RNA: Involvement of intramolecular hybrids in splicing of cob intron 4 RNA by mutation and reversion

Revertants have been obtained from six mutants of the box9 cluster, which are supposed to be defective in RNA splicing as a result of alterations in a splice signal sequence. This sequence is in the 5′ part of intron 4 of the cob gene, 330 to 340 bp downstream from the 5′ splice site. Sequencing reveals that reversion to splicing competence is achieved by restoration of the wild-type box9 sequence; by creation of novel box9 sequences; and by introduction of a second site or suppressor mutation ( sup −) compensating for the effect of the primary box9- mutation. The sup − mutation alters a sequence in intron 4, 293 bp upstream from the box9 − primary mutation. The box9 sequence and this upstream sequence can base pair to form an intramolecular hybrid in intron RNA in which box9 − and sup − are compensatory base pair exchanges (G → A and C → U, respectively). Thus intramolecular hybrid structures of intron RNA are essential for RNA splicing.