Antibiotic-induced Oligomerisation of Group I Intron RNA

Antibiotics act as inhibitors of various biological processes. Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs. The linear intron, which is excised during the self-splicing process, is still an active molecule capable of performing an intramolecular transesterification resulting in a circular molecule. However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers. The antibiotic stimulates the intron to reactin transinstead ofin cis. The phage T4-derivedtdintron uses the same sites for oligomerisation as for circularisation. Gel-retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic. It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA – RNA interactions and thus enabling their reaction. The tuberactinomycins are further able to induce the interaction of heterologous introns. The ligation of the T4 phage-derivedtdintron with theTetrahymenarRNA intron is very efficient, resulting in molecules composed of two introns derived from different species. Thetdintron attacks theTetraymenaintron at various sites, which are located within double-stranded regions. These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA–RNA interactions in a pre-protein era.     

Inhibition of In Vitro Splicing of a Group I Intron of Pneumocystis carinii

Unlike its mammalian hosts, the opportunistic fungal pathogen Pneumocystis carinii harbors group I self-splicing introns in its chromosomal genes encoding rRNA. This difference between pathogen and host suggests that intron splicing is a promising target for chemotherapy. We have found that intron splicing in vitro is inhibited by the anti- Pneumocystis agent pentamidine and by a series of pentamidine analogues, as well as by some aminoglycosides, tetracycline, L-arginine and ethidium bromide. Further studies will be needed to determine if this is the mechanism of action of pentamidine against P. carinii .     

Phylogeny and Self-Splicing Ability of the Plastid tRNA-Leu Group I Intron

Group I introns are mobile RNA enzymes (ribozymes) that encode conserved primary and secondary structures required for autocatalysis. The group I intron that interrupts the tRNA-Leu gene in cyanobacteria and plastids is remarkable because it is the oldest known intervening sequence and may have been present in the common ancestor of the cyanobacteria (i.e., 2.7–3.5 billion years old). This intron entered the eukaryotic domain through primary plastid endosymbiosis. We reconstructed the phylogeny of the tRNA-Leu intron and tested the in vitro self-splicing ability of a diverse collection of these ribozymes to address the relationship between intron stability and autocatalysis. Our results suggest that the present-day intron distribution in plastids is best explained by strict vertical transmission, with no intron losses in land plants or a subset of the Stramenopiles (xanthophyceae/phaeophyceae) and frequent loss among green algae, as well as in the red algae and their secondary plastid derivatives (except the xanthophyceae/phaeophyceae lineage). Interestingly, all tested land plant introns could not self-splice in vitro and presumably have become dependent on a host factor to facilitate in vivo excision. The host dependence likely evolved once in the common ancestor of land plants. In all other plastid lineages, these ribozymes could either self-splice or complete only the first step of autocatalysis. The first two authors (Dawn Simon and David Fewer) have contributed equally to this work. Present address (David Fewer): Department of Applied Chemistry and Microbiology, Viikki Biocenter, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Helsinki, Finland     

The Peperomia Mitochondrial coxI Group I Intron: Timing of Horizontal Transfer and Subsequent Evolution of the Intron

The Peperomia polybotrya coxI gene intron is the only currently reported group I intron in a vascular plant mitochondrial genome and it likely originated by horizontal transfer from a fungal donor. We provide a clearer picture of the horizontal transfer and a portrayal of the evolution of the group I intron since it was gained by the Peperomia mitochondrial genome. The intron was transferred recently in terms of plant evolution, being restricted to the single genus Peperomia among the order Piperales. Additional support is presented for the suggestion that a recombination/repair mechanism was used by the intron for integration into the Peperomia mitochondrial genome, as a perfect 1:1 correspondence exists between the intron's presence in a species and the presence of divergent nucleotide markers flanking the intron insertion site. Sequencing of coxI introns from additional Peperomia species revealed that several mutations have occurred in the intron since the horizontal transfer, but sequence alterations have not caused frameshifts or created stop codons in the intronic open reading frame. In addition, two coxI pseudogenes in Peperomia cubensis were discovered that lack a large region of coxI exon 2 and contain a truncated version of the group I intron that likely cannot be spliced out. Received: 29 May 1997 / Accepted: 1 November 1997     

Nested Evolution of a tRNALeu(UAA) Group I Intron by both Horizontal Intron Transfer and Recombination of the Entire tRNA Locus

The origin and evolution of bacterial introns are still controversial issues. Here we present data on the distribution and evolution of a recently discovered divergent tRNA(Leu)(UAA) intron. The intron shows a higher sequence affiliation with introns in tRNA(Ile)(CAU) and tRNA(Arg)(CCU) genes in alpha- and beta-proteobacteria, respectively, than with other cyanobacterial tRNA(Leu)(UAA) group I introns. The divergent tRNA(Leu)(UAA) intron is sporadically distributed both within the Nostoc and the Microcystis radiations. The complete tRNA gene, including flanking regions and intron from Microcystis aeruginosa strain NIVA-CYA 57, was sequenced in order to elucidate the evolutionary pattern of this intron. Phylogenetic reconstruction gave statistical evidence for different phylogenies for the intron and exon sequences, supporting an evolutionary model involving horizontal intron transfer. The distribution of the tRNA gene, its flanking regions, and the introns were addressed by Southern hybridization and PCR amplification. The tRNA gene, including the flanking regions, were absent in the intronless stains but present in the intron-containing strains. This suggests that the sporadic distribution of this intron within the Microcystis genus cannot be attributed to intron mobility but rather to an instability of the entire tRNA(Leu)(UAA) intron-containing genome region. Taken together, the complete data set for the evolution of this intron can best be explained by a model involving a nested evolution of the intron, i.e., wherein the intron has been transferred horizontally (probably through a single or a few events) to a tRNA(Leu)(UAA) gene which is located within a unstable genome region.     

Protein-Facilitated Folding of Group II Intron Ribozymes

Multiple studies hypothesize that DEAD-box proteins facilitate folding of the ai5γ group II intron. However, these conclusions are generally inferred from splicing kinetics, and not from direct monitoring of DEAD-box protein-facilitated folding of the intron. Using native gel electrophoresis and dimethyl sulfate structural probing, we monitored Mss-116-facilitated folding of ai5γ intron ribozymes and a catalytically active self-splicing RNA containing full-length intron and short exons. We found that the protein directly stimulates folding of these RNAs by accelerating formation of the compact near-native state. This process occurs in an ATP-independent manner, although ATP is required for the protein turnover. As Mss 116 binds RNA nonspecifically, most binding events do not result in the formation of the compact state, and ATP is required for the protein to dissociate from such nonproductive complexes and rebind the unfolded RNA. Results obtained from experiments at different concentrations of magnesium ions suggest that Mss 116 stimulates folding of ai5γ ribozymes by promoting the formation of unstable folding intermediates, which is then followed by a cascade of folding events resulting in the formation of the compact near-native state. Dimethyl sulfate probing results suggest that the compact state formed in the presence of the protein is identical to the near-native state formed more slowly in its absence. Our results also indicate that Mss 116 does not stabilize the native state of the ribozyme, but that such stabilization results from binding of attached exons.     

Patterns of Group I Intron Presence in Nuclear SSU rDNA of the Lichen Family Parmeliaceae

Group I introns are commonly reported within nuclear SSU ribosomal DNA of eukaryotic micro-organisms, especially in lichen-forming fungi. We have studied the primary and secondary structure of 70 new nuclear SSU rDNA group I introns of Parmeliaceae (Ascomycota: Lecanorales) and compared them with those available in databases, covering more than 60 species. The analyzed samples of Parmeliaceae fell into two groups, one having an intron at the 1506 site and another lacking this one but having another at the 1516 or 1521 position. Introns at the 1521 position seem to be transposed from 1516 sites. Introns at the 1516 position were similar in structure to ones previously reported at this site and known from other lecanoralean fungi, while those at the 1506 position showed structural differences and no similar introns are known from related fungi. The study of the distribution of group I introns within a large monophyletic ensemble of fungi has revealed an unexpected correlation between intron types and ecological and geographical parameters. The introns at the 1516 position occurred in mainly arctic, boreal, and temperate lichens, while those at position 1506 were present in mainly tropical and subtropical to oceanic mild-temperate taxa. Further, the 1516 introns occurred in genera with few distributed species that could represent older taxa, while the 1506 ones were mainly in species-rich genera that could be of recent speciation, as many species have wide distribution areas. The transition between two different environments has been accompanied by a change in introns gained and lost. [Reviewing Editor: Dr. Debashish Bhattacharya]     

A Group I Self-Splicing Intron in the Flagellin Gene of the Thermophilic Bacterium Geobacillus stearothermophilus

A group I intron that can be spliced in vivo and in vitro was identified in the flagellin gene of the thermophilic bacterium Geobacillus stearothermophilus. We also found one or two intervening sequences (IVS) of flagellin genes in five additional bacterial species. Furthermore, we report the presence of these sequences in two sites of a highly conserved region in the flagellin gene.     

An Unspliced Group I Intron in 23S rRNA Links Chlamydiales, Chloroplasts, and Mitochondria

Chlamydia was the only genus in the order Chlamydiales until the recent characterization of Simkania negevensis Z(T) and Parachlamydia acanthamoebae strains. The present study of Chlamydiales 23S ribosomal DNA (rDNA) focuses on a naturally occurring group I intron in the I-CpaI target site of 23S rDNA from S. negevensis. The intron, SnLSU. 1, belonged to the IB4 structural subgroup and was most closely related to large ribosomal subunit introns that express single-motif, LAGLIDADG endonucleases in chloroplasts of algae and in mitochondria of amoebae. RT-PCR and electrophoresis of in vivo rRNA indicated that the intron was not spliced out of the 23S rRNA. The unspliced 658-nt intron is the first group I intron to be found in bacterial rDNA or rRNA, and it may delay the S. negevensis developmental replication cycle by affecting ribosomal function.     

The Mitochondrial Genome of Acropora tenuis (Cnidaria; Scleractinia) Contains a Large Group I Intron and a Candidate Control Region

The complete nucleotide sequence of the mitochondrial genome of the coral Acropora tenuis has been determined. The 18,338 bp A. tenuis mitochondrial genome contains the standard metazoan complement of 13 protein-coding and two rRNA genes, but only the same two tRNA genes (trnM and trnW) as are present in the mtDNA of the sea anemone, Metridium senile. The A. tenuis nad5 gene is interrupted by a large group I intron which contains ten protein-coding genes and rns; M. senile has an intron at the same position but this contains only two protein-coding genes. Despite the large distance (about 11.5 kb) between the 5?-exon and 3?-exon boundaries, the A. tenuis nad5 gene is functional, as we were able to RT-PCR across the predicted intron splice site using total RNA from A. tenuis. As in M. senile, all of the genes in the A. tenuis mt genome have the same orientation, but their organization is completely different in these two zoantharians: The only common gene boundaries are those at each end of the group I intron and between trnM and rnl. Finally, we provide evidence that the rns-cox3 intergenic region in A. tenuis may correspond to the mitochondrial control region of higher animals. This region contains repetitive elements, and has the potential to form secondary structures of the type characteristic of vertebrate D-loops. Comparisons between a wide range of Acropora species showed that a long hairpin predicted in rns-cox3 is phylogenetically conserved, and allowed the tentative identification of conserved sequence blocks.     

A Group I Intron in the Nuclear Small Subunit rRNA Gene of Cryptendoxyla hypophloia, an Ascomycetous Fungus: Evidence for a New Major Class of Group I Introns

The ascomycetous fungus Cryptendoxyla hypophloia contains an insertion of 433 base pairs in the genes encoding nuclear small subunit ribosomal RNA. Secondary structure analyses of the insert reveal characteristics indicative of a Group I intron, including elements P, Q, R, and S; however, the sequences of these conserved regions deviate significantly from recognized consensus sequences for Group I introns. Principal-components analysis, based on 79 nucleotide positions from the conserved core sequences of 93 Group I introns, identified 17 introns similar to that of C. hypophloia. This grouping, which includes inserts from phylogenetically diverse organisms, cannot readily be classified in any previously recognized major group of Group I introns. We propose the creation of a new group, IE, to accommodate these sequences, and discuss the evolutionary relationships between group IE and other major groups of Group I introns. Received: 11 January 1998 / Accepted: 12 October 1998     

Group I Introns from Zygomycota: Evolutionary Implications for the Fungal IC1 Intron Subgroup

The origins of fungal group I introns within nuclear small-subunit (nSSU) rDNA are enigmatic. This is partly because they have never been reported in basal fungal phyla (Zygomycota and Chytridiomycota), which are hypothesized to be ancestral to derived phyla (Ascomycota and Basidiomycota). Here we report group I introns from the nSSU rDNA of two zygomycete fungi, Zoophagus insidians (Zoopagales) and Coemansia mojavensis (Kickxellales). Secondary structure analyses predicted that both introns belong to the IC1 subgroup and that they are distantly related to each other, which is also suggested by different insertion sites. Molecular phylogenetic analyses indicated that the IC1 intron of Z. insidians is closely related to the IC1 intron inserted in the LSU rDNA of the basidiomycete fungus Clavicorona taxophila, which strongly suggests interphylum horizontal transfer. The IC1 intron of C. mojavensis has a low phylogenetic affinity to other fungal IC1 introns inserted into site 943 of nSSU rDNA (relative to E. coli 16S rDNA). It is noteworthy that this intron contains a putative ORF containing a His–Cys box motif in the antisense strand, a hallmark for nuclear-encoded homing endonucleases. Overall, molecular phylogenetic analyses do not support the placement of these two introns in basal fungal IC1 intron lineages. This result leads to the suggestion that fungal IC1 introns might have invaded or been transferred laterally after the divergence of the four major fungal phyla. Received: 8 February 2001 / Accepted: 1 November 2001     

Evolutionary Dynamics of the mS952 Intron: A Novel Mitochondrial Group II Intron Encoding a LAGLIDADG Homing Endonuclease Gene

Examination of the mitochondrial small subunit ribosomal RNA (rns) gene of five species of the fungal genus Leptographium revealed that the gene has been invaded at least once at position 952 by a group II intron encoding a LAGLIDADG homing endonuclease gene. Phylogenetic analyses of the intron and homing endonuclease sequences indicated that each element in Leptographium species forms a single clade and is closely related to the group II intron/homing endonuclease gene composite element previously reported at position 952 of the mitochondrial rns gene of Cordyceps species and of Cryphonectria parasitica. The results of an intron survey of the mt rns gene of Leptographium species superimposed onto the phylogenetic analysis of the host organisms suggest that the composite element was transmitted vertically in Leptographium lundbergii. However, its stochastic distribution among strains of L. wingfieldii, L. terebrantis, and L. truncatum suggests that it has been horizontally transmitted by lateral gene transfer among these species, although the random presence of the intron may reflect multiple random loss events. A model is proposed describing the initial invasion of the group II intron in the rns gene of L. lundbergii by a LAGLIDADG homing endonuclease gene and subsequent evolution of this gene to recognize a novel DNA target site, which may now promote the mobility of the intron and homing endonuclease gene as a composite element.