Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution

Group I and group II introns are different catalytic self-splicing and mobile RNA elements that contribute to genome dynamics. In this study, we have analyzed their distribution and evolution in 29 sequenced genomes from the Bacillus cereus group of bacteria. Introns were of different structural classes and evolutionary origins, and a large number of nearly identical elements are shared between multiple strains of different sources, suggesting recent lateral transfers and/or that introns are under a strong selection pressure. Altogether, 73 group I introns were identified, inserted in essential genes from the chromosome or newly described prophages, including the first elements found within phages in bacterial plasmids. Notably, bacteriophages are an important source for spreading group I introns between strains. Furthermore, 77 group II introns were found within a diverse set of chromosomal and plasmidic genes. Unusual findings include elements located within conserved DNA metabolism and repair genes and one intron inserted within a novel retroelement. Group II introns are mainly disseminated via plasmids and can subsequently invade the host genome, in particular by coupling mobility with host cell replication. This study reveals a very high diversity and variability of mobile introns in B. cereus group strains.     

Origin and evolution of chloroplast group I introns in lichen algae

The history of group I introns is characterized by repeated horizontal transfers, even among phylogenetically distant species. The symbiogenetic thalli of lichens are good candidates for the horizontal transfer of genetic material among distantly related organisms, such as fungi and green algae. The main goal of this study was to determine whether there were different trends in intron distribution and properties among Chlorophyte algae based on their phylogenetic relationships and living conditions. Therefore, we investigated the occurrence, distribution and properties of group I introns within the chloroplast LSU rDNA in 87 Chlorophyte algae including lichen and free‐living Trebouxiophyceae compared to free‐living non‐Trebouxiophyceae species. Overall, our findings showed that there was high diversity of group I introns and homing endonucleases (HEs) between Trebouxiophyceae and non‐Trebouxiophyceae Chlorophyte algae, with divergence in their distribution patterns, frequencies and properties. However, the differences between lichen Trebouxiophyceae and free‐living Trebouxiophyceae were smaller. An exception was the cL2449 intron, which was closely related to ω elements in yeasts. Such introns seem to occur more frequently in lichen Trebouxiophyceae compared to free‐living Trebouxiophyceae. Our data suggest that lichenization and maintenance of lichen symbiosis for millions of years of evolution may have facilitated horizontal transfers of specific introns/HEs between symbionts. The data also suggest that sequencing of more chloroplast genes harboring group I introns in diverse algal groups may help us to understand the group I intron/HE transmission process within these organisms.     

Origin and evolution of group I introns in cyanobacterial tRNA genes.

Many tRNA(Leu)UAA genes from plastids contain a group I intron. An intron is also inserted in the same gene at the same position in cyanobacteria, the bacterial progenitors of plastids, suggesting an ancient bacterial origin for this intron. A group I intron has also been found in the tRNA(fMet) gene of some cyanobacteria but not in plastids, suggesting a more recent origin for this intron. In this study, we investigate the phylogenetic distributions of the two introns among cyanobacteria, from the earliest branching to the more derived species. The phylogenetic distribution of the tRNA(Leu)UAA intron follows the clustering of rRNA sequences, being either absent or present in clades of closely related species, with only one exception in the Pseudanabaena group. Our data support the notion that the tRNA(Leu)UAA intron was inherited by cyanobacteria and plastids through a common ancestor. Conversely, the tRNA(fMet) intron has a sporadic distribution, implying that many gains and losses occurred during cyanobacterial evolution. Interestingly, a phylogenetic tree inferred from intronic sequences clearly separates the different tRNA introns, suggesting that each family has its own evolutionary history.     

The evolution of homing endonuclease genes and group I introns in nuclear rDNA

Group I introns are autonomous genetic elements that can catalyze their own excision from pre-RNA. Understanding how group I introns move in nuclear ribosomal (r)DNA remains an important question in evolutionary biology. Two models are invoked to explain group I intron movement. The first is termed homing and results from the action of an intron-encoded homing endonuclease that recognizes and cleaves an intronless allele at or near the intron insertion site. Alternatively, introns can be inserted into RNA through reverse splicing. Here, we present the sequences of two large group I introns from fungal nuclear rDNA, which both encode putative full-length homing endonuclease genes (HEGs). Five remnant HEGs in different fungal species are also reported. This brings the total number of known nuclear HEGs from 15 to 22. We determined the phylogeny of all known nuclear HEGs and their associated introns. We found evidence for intron-independent HEG invasion into both homologous and heterologous introns in often distantly related lineages, as well as the "switching" of HEGs between different intron peripheral loops and between sense and antisense strands of intron DNA. These results suggest that nuclear HEGs are frequently mobilized. HEG invasion appears, however, to be limited to existing introns in the same or neighboring sites. To study the intron-HEG relationship in more detail, the S943 group I intron in fungal small-subunit rDNA was used as a model system. The S943 HEG is shown to be widely distributed as functional, inactivated, or remnant ORFs in S943 introns.     

Structure and assembly of group I introns

Self-splicing group I introns have served as a model for RNA catalysis and folding for over two decades. New three-dimensional structures now bring the details into view. Revelations include an unanticipated turn in the RNA backbone around the guanosine-binding pocket. Two metal ions in the active site coordinate the substrate and phosphates from all three helical domains.     

The antiquity of group I introns.

The recent discovery of self-splicing introns in cyanobacteria has given renewed interest to the question of whether introns may have been present in the ancestor of all living things. The properties of introns in genes of bacteria and bacteriophages are discussed in the context of their possible origin and biological function.     

Long-term evolution of the S788 fungal nuclear small subunit rRNA group I introns

More than 1000 group I introns have been identified in fungal rDNA. Little is known, however, of the splicing and secondary structure evolution of these ribozymes. Here, we use a combination of comparative and biochemical methods to address the evolution and splicing of a vertically inherited group I intron found at position 788 in the fungal small subunit (S) rRNA. The ancestral state of the S788 intron contains a highly conserved core and an extended P5 domain typical of IC1 introns. In contrast, the more derived introns have lost most of P5, and have an accelerated divergence rate within the core region with three functionally important substitutions that unambiguously separate them from the ancestral pool. Of 14 S788 group I introns that were tested for splicing, five, all of the ancestral type, were able to self-splice and produced intron RNA circles in vitro. The more derived S788 introns did not self-splice, and potentially rely on fungal-specific factors to facilitate splicing. In summary, we demonstrate one possible fate of vertically inherited group I introns, the loss of secondary structure elements, lessened selective constraints in the intron core, and ultimately, dependence on host-mediated splicing.     

The natural history of group I introns

There are four major classes of introns: self-splicing group I and group II introns, tRNA and/or archaeal introns and spliceosomal introns in nuclear pre-mRNA. Group I introns are widely distributed in protists, bacteria and bacteriophages. Group II introns are found in fungal and land plant mitochondria, algal plastids, bacteria and Archaea. Group II and spliceosomal introns share a common splicing pathway and might be related to each other. The tRNA and/or archaeal introns are found in the nuclear tRNA of eukaryotes and in archaeal tRNA, rRNA and mRNA. The mechanisms underlying the self-splicing and mobility of a few model group I introns are well understood. By contrast, the role of these highly distinct processes in the evolution of the 1500 group I introns found thus far in nature (e.g. in algae and fungi) has only recently been clarified. The explosion of new sequence data has facilitated the use of comparative methods to understand group I intron evolution in a broader context and to generate hypotheses about intron insertion, splicing and spread that can be tested experimentally.     

Structural conventions for group I introns.

Conventions for nomenclature of structural elements and a standard secondary structure representation for group I introns have been established by workers in the field. These conventions are designed to facilitate effective communication of information concerning the structure and function of these self-splicing introns.     

Identification of a family of group II introns encoding LAGLIDADG ORFs typical of group I introns

Group I and group II introns are unrelated classes of introns that each encode proteins that facilitate intron splicing and intron mobility. Here we describe a new subfamily of nine introns in fungi that are group II introns but encode LAGLIDADG ORFs typical of group I introns. The introns have fairly standard group IIB1 RNA structures and are inserted into three different sites in SSU and LSU rRNA genes. Therefore, introns should not be assumed to be group I introns based solely on the presence of a LAGLIDADG ORF.     

Atomic level architecture of group I introns revealed

Twenty-two years after their discovery as ribozymes, the self-splicing group I introns are finally disclosing their architecture at the atomic level. The crystal structures of three group I introns solved at moderately high resolution (3.1-3.8A) reveal a remarkably conserved catalytic core bound to the metal ions required for activity. The structure of the core is stabilized by an intron-specific set of long-range interactions that involves peripheral elements. Group I intron structures thus provide much awaited and extremely valuable snapshots of how these ribozymes coordinate substrate binding and catalysis.     

Molecular phylogeny of phyllopharyngean ciliates and their group I introns

We analyzed small subunit ribosomal DNA (ssu-rDNA) sequences to evaluate both the monophyly of the ciliate class Phyllopharyngea de Puytorac et al. (1974), and relationships among subclasses. Classifications based on morphology and ultrastructure divide the Phyllopharyngea into four subclasses, the Phyllopharyngia, Chonotrichia, Rhynchodia, and Suctoria. Our analyses of ssu-rDNA genealogies derived from sequence data collected from diverse members representing three of the four subclasses of Phyllopharyngea (Suctoria: Ephelota spp., Prodiscophyra collini, Acineta sp.; Phyllopharyngia: Chlamydodon exocellatus, Chlamydodon triquetrus, Dysteria sp.; and Chonotrichia: Isochona sp.) provide strong support for the monophyly of the Phyllopharyngea, and show that the Chonotrichia emerge from within the Phyllopharyngia. Based on this initial sampling, suctorian budding types are monophyletic, and exogenous budding appears to be basal to evaginative and endogenous budding. Further, we report the discovery of a group I intron at position 891 in the Suctoria Acineta sp. and Tokophrya lemnarum, and a second group I intron at position 1506 in T. lemnarum. These introns represent only the second examples of group I introns in a ciliate ribosomal gene, since the discovery of ribozymes in the LSU rRNA gene of Tetrahymena thermophila. Phylogenetic analyses of Group I introns suggest a complex evolutionary history involving either multiple loses or gains of introns within endogenously budding Suctoria.     

Toward predicting self-splicing and protein-facilitated splicing of group I introns

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.     

The molecular evolution and structural organization of self-splicing group I introns at position 516 in nuclear SSU rDNA of myxomycetes

Group I introns are relatively common within nuclear ribosomal DNA of eukaryotic microorganisms, especially in myxomycetes. Introns at position S516 in the small subunit ribosomal RNA gene are particularly common, but have a sporadic occurrence in myxomycetes. Fuligo septica, Badhamia gracilis, and Physarum flavicomum, all members of the family Physaraceae, contain related group IC1 introns at this site. The F. septica intron was studied at the molecular level and found to self-splice as naked RNA and to generate full-length intron RNA circles during incubation. Group I introns at position S516 appear to have a particularly widespread distribution among protists and fungi. Secondary structural analysis of more than 140 S516 group I introns available in the database revealed five different types of organization, including IC1 introns with and without His-Cys homing endonuclease genes, complex twin-ribozyme introns, IE introns, and degenerate group I-like introns. Both intron structural and phylogenetic analyses indicate a multiple origin of the S516 introns during evolution. The myxomycete introns are related to S516 introns in the more distantly related brown algae and Acanthamoeba species. Possible mechanisms of intron transfer both at the RNA- and DNA-levels are discussed in order to explain the observed widespread, but scattered, phylogenetic distribution.     

The molecular evolution and structural organization of group I introns at position 1389 in nuclear small subunit rDNA of myxomycetes

The number of nuclear group I introns from myxomycetes is rapidly increasing in GenBank as more rDNA sequences from these organisms are being sequenced. They represent an interesting and complex group of intervening sequences because several introns are mobile (or inferred to be mobile) and many contain large and unusual insertions in peripheral loops. Here we describe related group I introns at position 1389 in the small subunit rDNA of representatives from the myxomycete family Didymiaceae. Phylogenetic analyses support a common origin and mainly vertical inheritance of the intron. All S1389 introns from the Didymiaceae belong to the IC1 subclass of nuclear group I introns. The central catalytic core region of about 100 nt appears divergent in sequence composition even though the introns reside in closely related species. Furthermore, unlike the majority of group I introns from myxomycetes the S1389 introns do not self-splice as naked RNA in vitro under standard conditions, consistent with a dependence on host factors for folding or activity. Finally, the myxomycete S1389 introns are exclusively found within the family Didymiaceae, which suggests that this group I intron was acquired after the split between the families Didymiaceae and Physaraceae.     

New RNA-mediated reactions by yeast mitochondrial group I introns.

The group I self-splicing reaction is initiated by attack of a guanosine nucleotide at the 5' splice site of intron-containing precursor RNA. When precursor RNA containing a yeast mitochondrial group I intron is incubated in vitro under conditions of self-splicing, guanosine nucleotide attack can also occur at other positions: (i) the 3' splice site, resulting in formation of a 3' exon carrying an extra added guanosine nucleotide at its 5' end; (ii) the first phosphodiester bond in precursor RNA synthesized from the SP6 bacteriophage promoter, leading to substitution of the first 5'-guanosine by a guanosine nucleotide from the reaction mixture; (iii) the first phosphodiester bond in already excised intron RNA, resulting in exchange of the 5' terminal guanosine nucleotide for a guanosine nucleotide from the reaction mixture. An identical sequence motif (5'-GAA-3') occurs at the 3' splice site, the 5' end of SP6 precursor RNA and at the 5' end of excised intron RNA. We propose that the aberrant reactions can be explained by base-pairing of the GAA sequence to the Internal Guide Sequence. We suggest that these reactions are mediated by the same catalytic centre of the intron RNA that governs the normal splicing reactions.