Search

In vitro circular RNA (circRNA) preparation methods have been gaining a lot of attention recently as several reports suggest that circRNAs are more stable, with better performances in cells and in vivo, than linear RNAs in various biomedical applications. Self-splicing ribozymes are considered a major in vitro circRNA generation method for biomedical applications due to their simplicity and efficiency in the circularization of the gene of interest. This review summarizes, updates, and discusses the recently developed self-circularization methods based on the self-splicing ribozyme, such as group I and II intron ribozymes, and the pros and cons of each method in preparing circRNA in vitro. [ABSTRACT FROM AUTHOR]

The reproduction and dispersal strategies of lichens play a major role in shaping their population structure and photobiont diversity. Sexual reproduction, which is common, leads to high lichen genetic diversity and low photobiont selectivity. However, the lichen genus Endocarpon adopts a special co-dispersal model in which algal cells from the photobiont and ascospores from the mycobiont are released together into the environment. To explore the dispersal strategy impact on population structures, a total of 62 Endocarpon individuals and 12 related Verrucariaceae genera individuals, representing co-dispersal strategy and conventional independent dispersal mode were studied. Phylogenetic analysis revealed that Endocarpon, with a large-scale geographical distribution, showed an extremely high specificity of symbiotic associations with their photobiont. Furthermore, three types of group I intron at 1769 site have been found in most Endocarpon mycobionts, which showed a high variety of group I intron in the same insertion site even in the same species collected from one location. This study suggested that the ascospore-alga co-dispersal mode of Endocarpon resulted in this unusual mycobiont-photobiont relationship; also provided an evidence for the horizontal transfer of group I intron that may suggest the origin of the complexity and diversity of lichen symbiotic associations. [ABSTRACT FROM AUTHOR]

The current article describes Navicula vanseea sp. nov., a new species of diatom from Lake Van, a highly alkaline lake in Eastern Anatolia (Türkiye). The description is based on light and scanning electron microscopy performed on two monoclonal cultures. The complete nuclear rRNA clusters and plastid genomes have been sequenced for these two strains and the complete mitogenome for one of them. The plastome of both strains shows the probable loss of a functional ycf35 gene. They also exhibit two IB4 group I introns in their rrl, each encoding for a putative LAGLIDADG homing endonuclease, with the first L1917 IB4 intron reported amongst diatoms. The Maximum Likelihood phylogeny inferred from a concatenated alignment of 18S, rbcL and psbC distinguishes N. vanseea sp. nov. from the morphologically similar species Navicula cincta and Navicula microdigitoradiata. [ABSTRACT FROM AUTHOR]

Alternative splicing is an important mechanism in the process of eukaryotic nuclear mRNA precursors producing multiple protein products from a single gene. Although group I self-splicing introns usually perform regular splicing, limited examples of alternative splicing have also been reported. The exon-skipping type of splicing has been observed in genes containing two group I introns. To characterize splicing patterns (exon-skipping/exon-inclusion) of tandemly aligned group I introns, we constructed a reporter gene containing two Tetrahymena introns flanking a short exon. To control splicing patterns, we engineered the two introns in a pairwise manner to design pairs of introns that selectively perform either exon-skipping or exon-inclusion splicing. Through pairwise engineering and biochemical characterization, the structural elements important for the induction of exon-skipping splicing were elucidated. [ABSTRACT FROM AUTHOR]

Naturally occurring ribozymes with defined three-dimensional (3D) structures serve as promising platforms for the design and construction of artificial RNA nanostructures. We constructed a hexameric ribozyme nanostructure by face-to-face dimerization of a pair of triangular ribozyme trimers, unit RNAs of which were derived from the Tetrahymena group I ribozyme. In this study, we have expanded the dimerization strategy to a square-shaped ribozyme tetramer by introducing four pillar units. The resulting box-shaped nanostructures, which contained eight ribozyme units, can be assembled from either four or two components of their unit RNAs. [ABSTRACT FROM AUTHOR]

Vermamoeba vermiformis is a widespread free-living amoeba, playing a major role as a host of pathogens and probably acting as an opportunistic parasite. Identification is usually done by targeting the 18S rRNA gene which is highly conserved between different strains. In this study, the main sublines and subtypes of the amoeba were identified by molecular phylogeny of nearly complete sequences and by analysis of secondary structure. Complete analysis of the available sequences showed the presence of two heterologous introns in seven strains of Vermamoeba belonging to three distinct sublines. One strain has a conventional S788 intron, the others harbor an S943 intron with degenerated endonucleases. The two introns are phylogenetically related and show a common origin with an intron identified in Clandestinovirus, a giant virus infecting Vermamoeba. Current data suggests that these elements are part of a more diverse group that spreads horizontally in various hosts. [ABSTRACT FROM AUTHOR]

While current group I ribozymes use several distinct strategies to function under conditions of low Mg2+ concentration (≤ 3 mM), a deletion mutant of the Tetrahymena ribozyme (ΔP5 ribozyme) is virtually inactive with 3 mM Mg2+ due to removal of the large peripheral module, P5abc, supporting the active conformation of the core module. We investigated the molecular crowding effects of synthetic polyethylene glycols (PEGs) on the activity of the ΔP5 ribozyme. Among PEG molecules with different chain lengths, PEG600 improved the activity of the ΔP5 ribozyme most effectively in the presence of 3 mM Mg2+. [ABSTRACT FROM AUTHOR]

[Display omitted] • RNA structure-based approach reveals the intron landscape within human pathogenic fungi. • Group I introns are ubiquitously present in fungal pathogens, including all top-ranked human pathogens. • Representative introns from two major Candida pathogens catalyze highly efficient self-splicing reactions. Systemic fungal infections are a growing public health threat, and yet viable antifungal drug targets are limited as fungi share a similar proteome with humans. However, features of RNA metabolism and the noncoding transcriptomes in fungi are distinctive. For example, fungi harbor highly structured RNA elements that humans lack, such as self-splicing introns within key housekeeping genes in the mitochondria. However, the location and function of these mitochondrial riboregulatory elements has largely eluded characterization. Here we used an RNA-structure-based bioinformatics pipeline to identify the group I introns interrupting key mitochondrial genes in medically relevant fungi, revealing their fixation within a handful of genetic hotspots and their ubiquitous presence across divergent phylogenies of fungi, including all highest priority pathogens such as Candida albicans, Candida auris, Aspergillus fumigatus and Cryptococcus neoformans. We then biochemically characterized two representative introns from C. albicans and C. auris , demonstrating their exceptionally efficient splicing catalysis relative to previously-characterized group I introns. Indeed, the C. albicans mitochondrial intron displays extremely rapid catalytic turnover, even at ambient temperatures and physiological magnesium ion concentrations. Our results unmask a significant new set of players in the RNA metabolism of pathogenic fungi, suggesting a promising new type of antifungal drug target. [ABSTRACT FROM AUTHOR]

Ribozymes with modular structures are attractive platforms for the construction of nanoscale RNA objects with biological functions. We designed group I ribozyme dimers as unit ribozyme dimers (Urds), which self-assembled to form their polymeric states and also oligomeric states with defined numbers of Urds. Assembly of Urds yielded catalytic ability of a pair of distinct ribozyme units to cleave two distinct substrates. The morphologies of the assembled ribozyme structures were observed directly by atomic force microscopy (AFM). [ABSTRACT FROM AUTHOR]

Background: Increased contamination of European and Asian wheat and barley crops with "emerging" mycotoxins such as enniatins or beauvericin, produced by Fusarium avenaceum and Fusarium tricinctum, suggest that these phylogenetically close species could be involved in future food-safety crises. Results: The mitochondrial genomes of F. tricinctum strain INRA104 and F. avenaceum strain FaLH27 have been annotated. A comparative analysis was carried out then extended to a set of 25 wild strains. Results show that they constitute two distinct species, easily distinguished by their mitochondrial sequences. The mitochondrial genetic variability is mainly located within the intergenic regions. Marks of variations show they have evolved (i) by Single Nucleotide Polymorphisms (SNPs), (ii) by length variations mediated by insertion/deletion sequences (Indels), and (iii) by length mutations generated by DNA sliding events occurring in mononucleotide (A)n or (T)n microsatellite type sequences arranged in a peculiar palindromic organization. The optionality of these palindromes between both species argues for their mobility. The presence of Indels and SNPs in palindrome neighbouring regions suggests their involvement in these observed variations. Moreover, the intraspecific and interspecific variations in the presence/absence of group I introns suggest a high mobility, resulting from several events of gain and loss during short evolution periods. Phylogenetic analyses of intron orthologous sequences suggest that most introns could have originated from lateral transfers from phylogenetically close or distant species belonging to various Ascomycota genera and even to the Basidiomycota fungal division. Conclusions: Mitochondrial genome evolution between F. tricinctum and F. avenaceum is mostly driven by two types of mobile genetic elements, implicated in genome polymorphism. The first one is represented by group I introns. Indeed, both genomes harbour optional (inter- or intra-specifically) group I introns, all carrying putatively functional hegs, arguing for a high mobility of these introns during short evolution periods. The gain events were shown to involve, for most of them, lateral transfers between phylogenetically distant species. This study has also revealed a new type of mobile genetic element constituted by a palindromic arrangement of (A) n and (T) n microsatellite sequences whose presence was related to occurrence of SNPs and Indels in the neighbouring regions. [ABSTRACT FROM AUTHOR]

An intron landscape was prepared for the fungal mitochondrial nad5 gene. A hundred and eighty-eight fungal species were examined and a total of 265 introns were noted to be located in 29 intron insertion sites within the examined nad5 genes. Two hundred and sixty-three introns could be classified as group I types and two group II introns were noted. One additional group II intron module was identified nested within a composite group I intron. Based on features related to RNA secondary structures, introns can be classified into different subtypes and it was observed that intron insertion-sites are biased towards phase 0 and they appear to be specific to an intron type. Intron landscapes could be used as a guide map to predict the location of fungal mtDNA mobile introns, which are composite elements that include a ribozyme component and in some instances open reading frames encoding homing endonucleases or reverse transcriptases and all of these have applications in biotechnology. [ABSTRACT FROM AUTHOR]

Bimolecular ribozymes derived by physical dissection of unimolecular ribozymes consisting of two structural modules are promising platforms for the design and construction of assembled RNA nanostructures. Unit RNAs to be assembled intermolecularly into one-dimensional (1D) oligomers are designed by reconnecting the two structural modules in a manner different from the parent ribozymes. This strategy was applied to the Tetrahymena group I ribozyme. We constructed 1D ribozyme oligomers the assembly of which was observed by atomic force microscopy (AFM) and also controlled rationally to design a heterooctamer by differentiating the interface between the two modules. [ABSTRACT FROM AUTHOR]

Abstract Mitochondrial genome organization of sea anemones appears conserved among species and families, and is represented by a single circular DNA molecule of 17 to 21 kb. The mitochondrial gene content corresponds to the same 13 protein components of the oxidative phosphorylation (OxPhos) system as in vertebrates. Hallmarks, however, include a highly reduced tRNA gene repertoire and the presence of autocatalytic group I introns. Here we demonstrate that the mitochondrial genome of the deep-water sea anemone Protanthea simplex deviates significantly from that of other known sea anemones. The P. simplex mitochondrial genome contains a heavily scrambled order of genes that are coded on both DNA strands and organized along two circular mito-chromosomes, MCh-I and MCh-II. We found MCh-I to be representative of the prototypic sea anemone mitochondrial genome, encoding 12 OxPhos proteins, two ribosomal RNAs, two transfer RNAs, and a group I intron. In contrast, MCh-II was found to be a laterally transferred plasmid-like DNA carrying the conserved cytochrome oxidase II gene and a second allele of the small subunit ribosomal RNA gene. Highlights • The mtDNA consists of two circular mito-chromosomes; MCh-I and MCh-II. • MCh-I resembles a typical sea anemone mtDNA, but lacks the CO II gene. • MCh-I gene order is heavily rearranged and both DNA strands contain genes. • MCh-I contains a giant group I intron element of 15 kb within the ND5 gene. • MCh-II provides the CO II gene, and appears laterally transferred. [ABSTRACT FROM AUTHOR]

Tetrahymena mitochondrial cox1 barcodes and nuclear SSUrRNA sequences are particularly effective at distinguishing among its many cryptic species. In a project to learn more about Tetrahymena natural history, the majority of >1,000 Tetrahymena‐like fresh water isolates were assigned to established Tetrahymena species with the remaining assigned to 37 new species of Tetrahymena, nine new species of Dexiostoma and 12 new species of Glaucoma. Phylogenetically, all but three Tetrahymena species belong to the well‐established "australis" or "borealis" clades; the minority forms a divergent "paravorax" clade. Most Tetrahymena species are micronucleate, but others are exclusively amicronucleate. The self‐splicing intron of the LSUrRNA precursor is absent in Dexiostoma and Glaucoma and was likely acquired subsequent to the "australis/borealis" split; in some instances, its sequence is diagnostic of species. Tetrahymena americanis, T. elliotti, T. gruchyi n. sp., and T. borealis, together accounted for >50% of isolates, consistent with previous findings for established species. The biogeographic range of species found previously in Austria, China, and Pakistan was extended to the Nearctic; some species show evidence of population structure consistent with endemism. Most species were most frequently collected from ponds or lakes, while others, particularly Dexiostoma species, were collected most often from streams or rivers. The results suggest that perhaps hundreds of species remain to be discovered, particularly if collecting is global and includes hosts of parasitic forms. [ABSTRACT FROM AUTHOR]

Abstract In the RNA world, enrichment of self-replicating RNAs would have been beneficial to their survival, amplification, and evolution. Self-assembly of RNAs may be a strategy by which they enrich themselves. We examined the effects of molecular crowding on the activity of a bimolecular group I ribozyme and its derivative that self-assembles to form ribozyme oligomers. In a comparative activity assay using PEG as a molecular crowder, PEG rescued mutations in the parent bimolecular ribozyme more effectively than those in the oligomeric form. Highlights • PEG rescued mutations in the bimolecular group I ribozyme more effectively than those in the oligomeric form. [ABSTRACT FROM AUTHOR]

Abstract Among cationic molecules that can modulate ribozyme activities, polyamines act as both activator and inhibitor of ribozyme reactions partly due to their structural flexibility. Restriction of structural flexibility of polyamines may allow them to emphasize particular modulation effects. We examined eight stereoisomers of a synthetic pentamine bearing three cyclopentane rings. In the reaction of a structurally unstable group I ribozyme, three stereoisomers exhibited distinct effects as inhibitor, an additive with a neutral effect, and also as an activator. Highlights • Three stereoisomers of a pentamine showed distinct effects on a group I ribozyme. [ABSTRACT FROM AUTHOR]

Polyamines are a promising class of molecules that can modulate RNA enzyme activities. To analyze the effects of the number of amine moieties systematically, we employed four polyamines sharing dimethylene units to connect amine moieties. As a model RNA enzyme, we used a structurally unstable group I ribozyme, which was activated most and least efficiently by tetraethylenepentamine and diethylenetriamine respectively. [ABSTRACT FROM AUTHOR]

The mitochondrial genomes of sea anemones are dynamic in structure. Invasion by genetic elements, such as self-catalytic group I introns or insertion-like sequences, contribute to sea anemone mitochondrial genome expansion and complexity. By using next generation sequencing we investigated the complete mtDNAs and corresponding transcriptomes of the temperate sea anemone Anemonia viridis and its closer tropical relative Anemonia majano . Two versions of fused homing endonuclease gene ( HEG ) organization were observed among the Actiniidae sea anemones; in-frame gene fusion and pseudo-gene fusion. We provided support for the pseudo-gene fusion organization in Anemonia species, resulting in a repressed HEG from the COI-884 group I intron. orfA , a putative protein-coding gene with insertion-like features, was present in both Anemonia species. Interestingly, orfA and COI expression were significantly up-regulated upon long-term environmental stress corresponding to low seawater pH conditions. This study provides new insights to the dynamics of sea anemone mitochondrial genome structure and function. [ABSTRACT FROM AUTHOR]

Abstract Group I intron ribozymes share common core elements that form a three-dimensional structure responsible for their catalytic activity. This core structure is unstable without assistance from additional factors that stabilize its tertiary structure. We examined biogenic triamine and tetraamine and also their fragments for their abilities to stabilize a structurally unstable group I ribozyme, ΔP5 ribozyme, derived from the Tetrahymena group I intron ribozyme by deleting its large activator module. Biogenic triamine (spermidine) and tetraamine (spermine) efficiently activated the ΔP5 ribozyme under conditions where the ribozyme was virtually inactive. These observations suggested that polyamines are promising small molecule modulators to activate and possibly inhibit the core catalytic ability of group I ribozymes. Highlights • Biogenic triamine and tetraamine efficiently activated the ∆P5 ribozyme. • Spermine (tetraamine) modestly inhibited the full-length Tetrahymena ribozyme. • Polyamines are promising small molecule modulators to activate and possibly inhibit the core catalytic ability of group I ribozymes. [ABSTRACT FROM AUTHOR]

A bimolecular ribozyme consisting of a core ribozyme (ΔP5 RNA) and an activator module (P5abc RNA) has been used as a platform to design assembled RNA nanostructures. The tight and specific assembly between the P5abc and ΔP5 modules depends on two sets of intermodule interactions. The interface between P5abc and ΔP5 must be controlled when designing RNA nanostructures. To expand the repertoire of molecular recognition in the P5abc/ΔP5 interface, we modified the interface by replacing the parent tertiary interactions in the interface with artificial interactions. The engineered P5abc/ΔP5 interfaces were characterized biochemically to identify those suitable for nanostructure design. The new interfaces were used to construct 2Δ-square and 1Δ-array RNA nanostructures. [ABSTRACT FROM AUTHOR]

Complex group I introns represent hallmarks of hexacoral mitochondrial genomes (mtDNAs). These intron elements are expected to influence the gene organization and gene expression. We sequenced the mitochondrial genome and transcriptome of Zoanthus sansibariscus and Palythoa heliodiscus , two zoantharian species (colonial anemones) representing different families within the suborder Brachycnemina. The circular and approximately 21 kb mtDNAs contained two group I introns, one in ND5 and another in COI . The ND5-717 intron harbored two conventional mitochondrial genes ( ND1 and ND3 ) within its structure and revealed several conserved features compared to ND5-717 in sea anemones. The COI intron, however, was inserted at a unique location (after position 867), which was different from that in sea anemones (position 884) and stony corals (position 720). COI-867 contained a homing endonuclease gene ( HEG ) with remarkable features, including species-specific length variations and only one copy of the essential LAGLIDADG motif. Whereas transcriptome analysis indicated that all conventional mtDNA genes were expressed, HEG expression appeared significantly repressed. Finally, we identified absolutely conserved non-coding repeat motifs with antisense features and potential regulatory functions. [ABSTRACT FROM AUTHOR]

RNA is a biopolymer that is attractive for constructing nano-scale objects with complex structures. Threedimensional (3D) structures of naturally occurring RNAs often have modular architectures. The 3D structure of a group I (GI) ribozyme from Tetrahymena has a typical modular architecture, which can be separated into two structural modules ("P5 and P5abc). The fully active ribozyme can be reconstructed by assembling the two separately prepared modules through highly specific and strong assembly between "P5 ribozyme and P5abc RNA. Such non-covalent assembly of the two modules allows the design of polygonal RNA nano-structures. Through rational redesign of the parent GI ribozyme, we constructed variant GI ribozymes as unit RNAs for polygonal-shaped (closed) oligomers with catalytic activity. Programmed trimerization and tetramerization of the unit RNAs afforded catalytically active nano-sized RNA triangles and squares, the structures of which were directly observed by atomic force microscopy (AFM). [ABSTRACT FROM AUTHOR]

Free-living amoebae of the genus Acanthamoeba are worldwide present in natural and artificial environments, and are also clinically important, as causative agents of diseases in humans and other animals. Acanthamoeba comprises several species, historically assigned to one of the three groups based on their cyst morphology, but presently recognized as at least 20 genotypes (T1-T20) on the basis of their nuclear 18S ribosomal RNA (rRNA) gene (18S rDNA) sequences. While strain identification may usually be achieved targeting short (<500 bp) 18S ribosomal DNA (rDNA) fragments, the use of full-length gene sequences (>2200 bp) is necessary for correct genotype description and reliable molecular phylogenetic inference. The genotype T15, corresponding to Acanthamoeba jacobsi, is the only genotype described on the basis of partial sequences (~1500 bp). While this feature does not prevent the correct identification of the strains, having only partial sequences renders the genotype T15 not completely defined and may furthermore affect its position in the Acanthamoeba molecular tree. Here, we complete this gap, by obtaining full-length 18S rDNA sequences from eight A. jacobsi strains, genotype T15. Morphologies and physiological features of isolated strains are reported. Molecular phylogeny based on full 18S rDNA confirms some previous suggestions for a genetic link between T15 and T13, T16, and T19, with T19 as sister-group to T15. [ABSTRACT FROM AUTHOR]

Background: Mitochondrial introns intermit coding regions of genes and feature characteristic secondary structures and splicing mechanisms. In metazoans, mitochondrial introns have only been detected in sponges, cnidarians, placozoans and one annelid species. Within demosponges, group I and group II introns are present in six families. Based on different insertion sites within the cox1 gene and secondary structures, four types of group I and two types of group II introns are known, which can harbor up to three encoding homing endonuclease genes (HEG) of the LAGLIDADG family (group I) and/or reverse transcriptase (group II). However, only little is known about sponge intron mobility, transmission, and origin due to the lack of a comprehensive dataset. We analyzed the largest dataset on sponge mitochondrial group I introns to date: 95 specimens, from 11 different sponge genera which provided novel insights into the evolution of group I introns. Results: For the first time group I introns were detected in four genera of the sponge family Scleritodermidae (Scleritoderma, Microscleroderma, Aciculites, Setidium). We demonstrated that group I introns in sponges aggregate in the most conserved regions of cox1. We showed that co-occurrence of two introns in cox1 is unique among metazoans, but not uncommon in sponges. However, this combination always associates an active intron with a degenerating one. Earlier hypotheses of HGT were confirmed and for the first time VGT and secondary losses of introns conclusively demonstrated. Conclusion: This study validates the subclass Spirophorina (Tetractinellida) as an intron hotspot in sponges. Our analyses confirm that most sponge group I introns probably originated from fungi. DNA barcoding is discussed and the application of alternative primers suggested. [ABSTRACT FROM AUTHOR]

Group I introns in nuclear ribosomal RNA of eukaryotic microorganisms are processed by splicing or circularization. The latter results in formation of full-length circular introns without ligation of the exons and has been proposed to be active in intron mobility. We applied qRT-PCR to estimate the copy number of circular intron RNA from the myxomycete Didymium iridis. In exponentially growing amoebae, the circular introns are nuclear and found in 70 copies per cell. During heat-shock, the circular form is up-regulated to more than 500 copies per cell. The intron harbours two ribozymes that have the potential to linearize the circle. To understand the structural features that maintain circle integrity, we performed chemical and enzymatic probing of the splicing ribozyme combined with molecular modeling to arrive at models of the inactive circular form and its active linear counterpart. We show that the two forms have the same overall structure but differ in key parts, including the catalytic core element P7 and the junctions at which reactions take place. These differences explain the relative stability of the circular species, demonstrate how it is prone to react with a target molecule for circle integration and thus supports the notion that the circular form is a biologically significant molecule possibly with a role in intron mobility. [ABSTRACT FROM AUTHOR]

Structured RNAs fold through multiple pathways, but we have little understanding of the molecular features that dictate folding pathways and determine rates along a given pathway. Here, we asked whether folding of a complex RNA can be understood from its structural modules. In a two-piece version of the Tetrahymena group I ribozyme, the separated P5abc subdomain folds to local native secondary and tertiary structure in a linked transition and assembles with the ribozyme core via three tertiary contacts: a kissing loop (P14), a metal core–receptor interaction, and a tetraloop–receptor interaction, the first two of which are expected to depend on native P5abc structure from the local transition. Native gel, NMR, and chemical footprinting experiments showed that mutations that destabilize the native P5abc structure slowed assembly up to 100-fold, indicating that P5abc folds first and then assembles with the core by conformational selection. However, rate decreases beyond 100-fold were not observed because an alternative pathway becomes dominant, with nonnative P5abc binding the core and then undergoing an induced-fit rearrangement. P14 is formed in the rate-limiting step along the conformational selection pathway but after the rate-limiting step along the induced-fit pathway. Strikingly, the assembly rate along the conformational selection pathway resembles that of an isolated kissing loop similar to P14, and the rate along the induced-fit pathway resembles that of an isolated tetraloop–receptor interaction. Our results indicate substantial modularity in RNA folding and assembly and suggest that these processes can be understood in terms of underlying structural modules. [ABSTRACT FROM AUTHOR]

Chloroplasts play an essential role in plant growth and development through manipulating photosynthesis and the production of hormones and metabolites. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components is still lacking. We isolated a rice (Oryza sativa) mutant, termed albino leaf 2 (al2), using genetic screening. Phenotypic analysis revealed that the al2 mutation caused obvious albino leaves at the early developmental stage, eventually leading to al2 seedling death. Electron microscopy investigations indicated that the chloroplast structure was disrupted in the al2 mutants at an early developmental stage and subsequently resulted in the breakdown of the entire chloroplast. Molecular cloning illustrated that AL2 encodes a chloroplast group IIA intron splicing facilitator (CRS1) in rice, which was confirmed by a genetic complementation experiment. Moreover, our results demonstrated that AL2 was constitutively expressed in various tissues, including green and non-green tissues. Interestingly, we found that the expression levels of a subset of chloroplast genes that contain group IIA and IIB introns were significantly reduced in the al2 mutant compared to that in the wild type, suggesting that AL2 is a functional CRS1 in rice. Differing from the orthologous CRS1 in maize and Arabidopsis that only regulates splicing of the chloroplast group II intron, our results demonstrated that the AL2 gene is also likely to be involved in the splicing of the chloroplast group I intron. They also showed that disruption of AL2 results in the altered expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded polymerases and nuclear-encoded chloroplast genes. Taken together, these findings shed new light on the function of nuclear-encoded chloroplast group I and II intron splicing factors in rice. [ABSTRACT FROM AUTHOR]

Background: Self-splicing introns are present in the mitochondria of members of most eukaryotic lineages. They are divided into Group I and Group II introns, according to their secondary structure and splicing mechanism. Being rare in animals, self-splicing introns were only described in a few sponges, cnidarians, placozoans and one annelid species. In sponges, three types of mitochondrial Group I introns were previously described in two demosponge families (Tetillidae, and Aplysinellidae) and in the homoscleromorph family Plakinidae. These three introns differ in their insertion site, secondary structure and in the sequence of the LAGLIDADG gene they encode. Notably, no group II introns have been previously described in sponges. Results: We report here the presence of mitochondrial introns in the cytochrome oxidase subunit 1 (COI) gene of three additional sponge species from three different families: Agelas oroides (Agelasidae, Agelasida), Cymbaxinellap verrucosa (Hymerhabdiidae, Agelasida) and Axinella polypoides (Axinellidae, Axinellida). We show, for the first time, that sponges can also harbour Group II introns in their COI gene, whose presence in animals' mitochondria has sofar been described in only two phyla, Placozoa and Annelida. Surprisingly, two different Group II introns were discovered in the COI gene of C. verrucosa. Phylogenetic analysis indicates that the Group II introns present in C. verrucosa are related to red algae (Rhodophyta) introns. Conclusions: The differences found among intron secondary structures and the phylogenetic inferences support the hypothesis that the introns originated from independent horizontal gene transfer events. Our results thus suggest that self-splicing introns are more diverse in the mitochondrial genome of sponges than previously anticipated. [ABSTRACT FROM AUTHOR]

Plasmodial slime molds are members of the class Amoebozoa forming elaborate fruit bodies releasing airborne spores. Two species concepts have been developed independently: a morphological relying on fruit body characters, and a biological relying on crossing studies of a few cultivable species. In an attempt to reconcile both concepts, we obtained for 198 specimens of the common species Trichia varia partial sequences of three independent markers (nuclear small-subunit (SSU) ribosomal RNA gene, extrachromosomal; elongation factor 1 alpha gene, chromosomal; cytochrome oxidase subunit 1 gene, mitochondrial). The resulting phylogeny revealed 21 three-marker genotypes clustering into three groups. Combinations of the single-marker genotypes occurred exclusively within these groups, called 1, 2a, and 2b. To examine the suitability of group I introns to monitor speciation events, complete SSU sequences were generated for 66 specimens, which revealed six positions that can carry group I introns. For each of the groups 1 and 2a, five of these positions were occupied by different intron genotypes; and no genotype was shared by the two groups. Group 2b was devoid of introns. Putatively functional or degenerated homing endonuclease genes were found at different positions in groups 1 and 2a. All observations (genotypic combinations of the three markers, signs of recombination, intron patterns) fit well into a pattern of three cryptic biological species that reproduce predominantly sexual but are reproductively isolated. The pattern of group I introns and inserted homing endonuclease genes mounts evidence that the Goddard-Burt intron life cycle model applies to naturally occurring myxomycete populations. [ABSTRACT FROM AUTHOR]

Two phagotrophic euglenid strains (Strains Pac and Tam) were isolated from coastal locations in Taiwan. Ultrastructural characteristics of the strains included five pellicle strips joined at the posterior end. The strips were formed by major grooves with bifurcated edges. At the cell anterior, the feeding structure formed a lip. Underneath the lip was a comb composed of layers of microtubules. Farther back, two supporting rods tapered toward the posterior end, and a number of vanes with attached microtubules were present between the rods. The morphological characteristics agree with Ploeotia costata Strain CCAP 1265/1. However, the 18S r DNA sequences of Strains Pac/Tam lacked a group I intron and possessed three extra insertions of 116, 67, and 53 bp. Phylogenetic analysis indicated low sequence similarity between Strains Pac/Tam and CCAP 1265/1 (92%). The morphospecies P. costata apparently includes a substantial level of DNA sequence divergence, and likely represents multiple molecular species units. [ABSTRACT FROM AUTHOR]

Sclerotinia trifoliorum, an important pathogen of cool season legumes, displays both homothallism and heterothallism in its life cycle, unique among members of the genus Sclerotinia. Very little is known about its genetic diversity and population structure. A sample of 129 isolates of S. trifoliorum from diseased chickpea in California was investigated for genetic diversity, population differentiation and reproductive mode. Genetic diversity was estimated using mycelial compatibility (MCG) phenotypes, rDNA intron variation, and allelic diversity at seven microsatellite loci. Genetic analysis revealed high levels of genotypic diversity demonstrated by high genotypic richness (0·88). Similarly, high levels of gene diversity (mean expected heterozygosity HE = 0·68) were observed at the microsatellite loci. Geographic populations of S. trifoliorum were highly admixed as evident from low FST values (0-0·11), suggesting high contemporary or historical gene flow. Hierarchical analysis of molecular variance showed that more than 92% of the genetic variation occurred among isolates within populations. Bayesian clustering analysis identified four cryptic genetic populations that were not correlated to geographic location, and index of multilocus association was non-significant in each of the four genetic populations. However, the presence of identical haplotypes within and among populations indicates clonal reproduction. The high levels of haplotype diversity and population heterogeneity, a lack of correspondence between MCG and microsatellite haplotype, and low levels of population differentiation suggest that populations of S. trifoliorum in chickpea have been undergoing extensive outcrossing and migration events probably shaped by human-mediated dissemination, the underlying diverse cropping systems, and chickpea disease management practices. [ABSTRACT FROM AUTHOR]

Introduction Approximately 100 million confirmed infections and 20,000 deaths are caused by Dengue virus (DENV) outbreaks annually. Global warming and rapid dispersal have resulted in DENV epidemics in formally non-endemic regions. Currently no consistently effective preventive measures for DENV exist, prompting development of transgenic and paratransgenic vector control approaches. Production of transgenic mosquitoes refractory for virus infection and/or transmission is contingent upon defining antiviral genes that have low probability for allowing escape mutations, and are equally effective against multiple serotypes. Previously we demonstrated the effectiveness of an anti-viral group I intron targeting U143 of the DENV genome in mediating trans-splicing and expression of a marker gene with the capsid coding domain. In this report we examine the effectiveness of coupling expression of ▵ N Bax to trans-splicing U143 intron activity as a means of suppressing DENV infection of mosquito cells. Results Targeting the conserved DENV circularization sequence (CS) by U143 intron trans-splicing activity appends a 3' exon RNA encoding ▵ N Bax to the capsid coding region of the genomic RNA, resulting in a chimeric protein that induces premature cell death upon infection. TCID50-IFA analyses demonstrate an enhancement of DENV suppression for all DENV serotypes tested over the identical group I intron coupled with the non-apoptotic inducing firefly luciferase as the 3' exon. These cumulative results confirm the increased effectiveness of this αDENV-U143-▵ N Bax group I intron as a sequence specific antiviral that should be useful for suppression of DENV in transgenic mosquitoes. Annexin V staining, caspase 3 assays, and DNA ladder observations confirm DCA-▵ N Bax fusion protein expression induces apoptotic cell death. Conclusion This report confirms the relative effectiveness of an anti-DENV group I intron coupled to an apoptosis-inducing ▵ N Bax 3' exon that trans-splices conserved sequences of the 5' CS region of all DENV serotypes and induces apoptotic cell death upon infection. Our results confirm coupling the targeted ribozyme capabilities of the group I intron with the generation of an apoptosis-inducing transcript increases the effectiveness of infection suppression, improving the prospects of this unique approach as a means of inducing transgenic refractoriness in mosquitoes for all serotypes of this important disease. [ABSTRACT FROM AUTHOR]

Abstract: Like many structured RNAs, the Tetrahymena group I intron ribozyme folds through multiple pathways and intermediates. Under standard conditions in vitro, a small fraction reaches the native state (N) with k obs ≈0.6min−1, while the remainder forms a long-lived misfolded conformation (M) thought to differ in topology. These alternative outcomes reflect a pathway that branches late in folding, after disruption of a trapped intermediate (Itrap). Here we use catalytic activity to probe the folding transitions from Itrap to the native and misfolded states. We show that mutations predicted to weaken the core helix P3 do not increase the rate of folding from Itrap but they increase the fraction that reaches the native state rather than forming the misfolded state. Thus, P3 is disrupted during folding to the native state but not to the misfolded state, and P3 disruption occurs after the rate-limiting step. Interestingly, P3-strengthening mutants also increase native folding. Additional experiments show that these mutants are rapidly committed to folding to the native state, although they reach the native state with approximately the same rate constant as the wild-type ribozyme (~1min−1). Thus, the P3-strengthening mutants populate a distinct pathway that includes at least one intermediate but avoids the M state, most likely because P3 and the correct topology are formed early. Our results highlight multiple pathways in RNA folding and illustrate how kinetic competitions between rapid events can have long-lasting effects because the “choice” is enforced by energy barriers that grow larger as folding progresses. [Copyright &y& Elsevier]

Highlights: [•] We present genomic resources of cold-water sea anemones. [•] An obligatory group I intron contains truncated duplicated gene sequences. [•] A mobile group I intron contains an expressed homing endonuclease gene. [•] We detect a novel insertion element that contains expressed protein coding genes. [•] Introns and insertion element have higher substitution rates. [Copyright &y& Elsevier]

Highlights: [•] The Chaetomium thermophilum mtDNA rns gene is interrupted by a twintron. [•] The external intron encoded homing endonuclease gene is interrupted by a group II intron. [•] In vitro RNA splicing assays shows that the group II intron can self-splice. [•] The mS1247 twintron encoded homing endonuclease is active and cuts within the rns gene. [•] This twintron has the potential to be developed into a genome editing tool. [ABSTRACT FROM AUTHOR]

Homing endonucleases are highly specific DNA cleaving enzymes that are encoded within genomes of all forms of microbial life including phage and eukaryotic organelles. These proteins drive the mobility and persistence of their own reading frames. The genes that encode homing endonucleases are often embedded within self-splicing elements such as group I introns, group II introns and inteins. This combination of molecular functions is mutually advantageous: the endonuclease activity allows surrounding introns and inteins to act as invasive DNA elements, while the splicing activity allows the endonuclease gene to invade a coding sequence without disrupting its product. Crystallographic analyses of representatives from all known homing endonuclease families have illustrated both their mechanisms of action and their evolutionary relationships to a wide range of host proteins. Several homing endonucleases have been completely redesigned and used for a variety of genome engineering applications. Recent efforts to augment homing endonucleases with auxiliary DNA recognition elements and/or nucleic acid processing factors has further accelerated their use for applications that demand exceptionally high specificity and activity. [ABSTRACT FROM AUTHOR]

Background: The availability of mitochondrial genomes has allowed for the resolution of numerous questions regarding the evolutionary history of fungi and other eukaryotes. In the Gibberella fujikuroi species complex, the exact relationships among the so-called "African", "Asian" and "American" Clades remain largely unresolved, irrespective of the markers employed. In this study, we considered the feasibility of using mitochondrial genes to infer the phylogenetic relationships among Fusarium species in this complex. The mitochondrial genomes of representatives of the three Clades (Fusarium circinatum, F. verticillioides and F. fujikuroi) were characterized and we determined whether or not the mitochondrial genomes of these fungi have value in resolving the higher level evolutionary relationships in the complex. Results: Overall, the mitochondrial genomes of the three species displayed a high degree of synteny, with all the genes (protein coding genes, unique ORFs, ribosomal RNA and tRNA genes) in identical order and orientation, as well as introns that share similar positions within genes. The intergenic regions and introns generally contributed significantly to the size differences and diversity observed among these genomes. Phylogenetic analysis of the concatenated protein-coding dataset separated members of the Gibberella fujikuroi complex from other Fusarium species and suggested that F. fujikuroi ("Asian" Clade) is basal in the complex. However, individual mitochondrial gene trees were largely incongruent with one another and with the concatenated gene tree, because six distinct phylogenetic trees were recovered from the various single gene datasets. Conclusion: The mitochondrial genomes of Fusarium species in the Gibberella fujikuroi complex are remarkably similar to those of the previously characterized Fusarium species and Sordariomycetes. Despite apparently representing a single replicative unit, all of the genes encoded on the mitochondrial genomes of these fungi do not share the same evolutionary history. This incongruence could be due to biased selection on some genes or recombination among mitochondrial genomes. The results thus suggest that the use of individual mitochondrial genes for phylogenetic inference could mask the true relationships between species in this complex. [ABSTRACT FROM AUTHOR]

Although black corals inhabit all the world's oceans, they have been relatively understudied as ∼185 of 247 species occur at depths > 50 m. Antipatharians have been included in several phylogenetic studies; however, sample sizes are small and taxonomic coverage minimal. Low levels of mitochondrial (mt) sequence divergence within Scleractinia and Octocorallia are assumed to apply to all anthozoans, although no formal study has been conducted on the order Antipatharia. To quantify genetic variation in the black coral mitogenome, we analysed DNA sequences of the two longest intergenic regions ( IGRs) and cox3-cox1 for 26 of 41 genera, representing all families and subfamilies. We also quantified divergence at the intraspecific level using six mtIGRs and their flanking protein-coding genes and rRNA for 100+ colonies of Antipathes griggi. Utilizing sequence data from the two mtIGRs, cox3-cox1, as well as nuclear 18S and 28S, we constructed the first multi-locus phylogenies of the Antipatharia. Reconstructions revealed that species in the genus Stichopathes are split across two families, Sibopathes macrospina groups among North Atlantic Parantipathes (suggesting the actinopharynx and mesenteries were secondarily lost), and three families are polyphyletic. These and other results provide novel, independent insights into the evolutionary history of antipatharians and support placement of species into higher-level groupings based on microscopic skeletal features rather than gross colony morphology. An illustrated key to the seven currently recognized families is also provided. © 2013 The Linnean Society of London [ABSTRACT FROM AUTHOR]

Abstract: RNAs are prone to misfolding, but how misfolded structures are formed and resolved remains incompletely understood. The Tetrahymena group I intron ribozyme folds in vitro to a long-lived misfolded conformation (M) that includes extensive native structure but is proposed to differ in topology from the native state (N). A leading model predicts that exchange of the topologies requires unwinding of the long-range, core helix P3, despite the presence of P3 in both conformations. To test this model, we constructed 16 mutations to strengthen or weaken P3. Catalytic activity and in-line probing showed that nearly all of the mutants form the M state before folding to N. The P3-weakening mutations accelerated refolding from M (3- to 30-fold) and the P3-strengthening mutations slowed refolding (6- to 1400-fold), suggesting that P3 indeed unwinds transiently. Upon depletion of Mg2+, the mutations had analogous effects on unfolding from N to intermediates that subsequently fold to M. The magnitudes for the P3-weakening mutations were larger than in refolding from M, and small-angle X-ray scattering showed that the ribozyme expands rapidly to intermediates from which P3 is disrupted subsequently. These results are consistent with previous results indicating unfolding of native peripheral structure during refolding from M, which probably permits rearrangement of the core. Together, our results demonstrate that exchange of the native and misfolded conformations requires loss of a core helix in addition to peripheral structure. Further, the results strongly suggest that misfolding arises from a topological error within the ribozyme core, and a specific topology is proposed. [Copyright &y& Elsevier]

Abstract: At 135,005nt, the mitochondrial genome in Agaricus bisporus represents the largest fungal mitochondrial genome sequenced to date. Its large size is mainly due to the presence of mobile genetic elements, including a total of 43 group I introns, three group II introns, and five DNA fragments that show sequence similarity to linear invertron-like plasmids. The introns are distributed in eight of the 15 protein coding genes. These introns contain a total of 61,092nt (∼45.3% of the whole mitochondrial genome) and include representatives of most of the group I introns so far found in mitochondrial genomes of Basidiomycota. The plasmid-like sequences include 6730nt total representing 5.0% of the genome. These sequences showed high-level similarities to two different mitochondrial plasmids reported for basidiomycete mushrooms: the autonomously replicating pEM in Agaricus bitorquis and the integrated linear plasmid sequences in Agrocybe aegerita and Moniliophthora perniciosa. Moreover, the plasmid-related sequences are located within or adjacent to two large (4559nt) inverted repeats containing also two sets of mitochondrial tRNA genes. Our analyses are consistent with the hypothesis that horizontal DNA transfer has played a significant role in the evolution of the A. bisporus mitochondrial genome. [Copyright &y& Elsevier]

Chlorella Beijerinck ( Chlorellaceae, Trebouxiophyceae) strains from the collection of the National Institute for Environmental Studies ( NIES) were characterized using gene sequence data. The misidentification of a number of strains was rectified. Chlorella vulgaris Beijerinck NIES-2173 was reclassified as C. sorokiniana Shihira et Krauss. Chlorella sp. NIES-2171 was described as a new species in the genus Micractinium Fresinius, M. inermum Hoshina et Fujiwara. Chlorella sorokiniana NIES-2167 and Chlorella sp. NIES-2330 were found to be phylogenetically related to Didymogenes Schmidle. We propose these two strains be transferred to the genus Didymogenes and given new names: D. sphaerica Hoshina et Fujiwara and D. soliella Hoshina et Fujiwara. Taxonomic decisions were primarily based on small subunit-internal transcribed spacer ribosomal DNA phylogeny for genus assignment and ITS2 sequence-structure to determine species autonomy. Our findings suggest that this strategy is the most effective way to use the species concept among autosporic coccoids. [ABSTRACT FROM AUTHOR]

Teleki rootstocks are used in grapevine-producing countries all over the world. They represent one of the largest groups of available rootstocks but their origin is still in dispute although they have been regarded as Vitis berlandieri × V. riparia hybrids. To investigate their possible origin, we amplified and sequenced three chloroplast regions, two non-coding spacers ( trnL-F, trnS-G) and the trnL group I intron in a core collection of Teleki rootstocks representing widespread accessions and related wild North American grape species ( V. berlandieri, V. riparia and V. rupestris). Concatenated sequence data coupled with microstructural changes discovered in the chloroplast regions provided data to trace the maternal ancestry of the Teleki lines. All chloroplast regions showed both nucleotide and length variation. Length mutations in the non-coding regions represented mostly simple sequence repeats of poly-A and -T stretches. These indel characters exhibited additional diversity comparable with the nucleotide diversity and increased resolution of the phylogenetic trees. We found that a group of Teleki accessions position together with the wild grape species V. riparia. Another group of Teleki rootstocks formed a sister group to the other North American species V. berlandieri. These clades had moderate support values, and they do not share ancestry with other accessions of Teleki rootstocks resolved with high support value in the V. riparia clade. It seems that Teleki-Kober 5BB and 125 AA accessions might have a V. berlandieri maternal background. We also found great differences within putative clones of Teleki 5C and Teleki-Kober 5BB suggesting that the selection of these accessions was performed on heterogenous or mislabeled plant material collectively maintained under these names. [ABSTRACT FROM AUTHOR]

Quinol oxidation inhibitors (QoIs) are one of the most important classes of fungicides used in agriculture. They block electron transfer between cytochrome b and cytochrome c1, thereby impeding the production of ATP via oxidative phosphorylation. QoI fungicides are generally at high risk of provoking resistance in fungal phytopathogens. Resistance has been reported in more than thirty species, amongst others, in Botrytis cinerea. In various QoI-resistant monosporic B. cinerea isolates from Hungary, a G-to-C point mutation was identified in the mitochondrial gene that encodes the QoI target, cytochrome b, resulting in a glycine to alanine substitution at position 143 (G143A). Analysis of Hungarian group I and group II strains further indicated the frequent occurrence of an additional group I-type intron in the cytb gene directly downstream of the glycine-143 codon. Mutual presence of distinct mitochondrial DNAs specifying different cytb alleles (heteroplasmy) has also been detected in monosporic strains. Remarkably, a number of group II field isolates were found to be highly resistant to azoxystrobin although they did not appear to carry the G-to-C mutation (G143A) generally associated with fungal QoI-resistance. [ABSTRACT FROM AUTHOR]

Arbuscular mycorrhizal fungi (AMF) are ubiquitous organisms that benefit ecosystems through the establishment of an association with the roots of most plants: the mycorrhizal symbiosis. Despite their ecological importance, however, these fungi have been poorly studied at the genome level. [ABSTRACT FROM AUTHOR]

Abstract: AIMP2/p38 is a scaffolding protein critical for the assembly of the macromolecular tRNA synthetase complex. Moreover, AIMP2 harbors anti-proliferative activity and promotes cell death as a proapototic factor. A splicing variant of AIMP2 lacking exon 2 (AIMP2-DX2) is specifically generated by an alternative splicing process and is highly expressed in human lung cancer cells and the tissues of cancer patients. AIMP2-DX2 induces tumorigenesis by compromising the tumor suppressor function of normal AIMP2. Here, we describe a novel approach to cancer therapy that is based on trans-splicing ribozyme-mediated replacement of specific RNAs. We developed a specific ribozyme that can target and replace the splicing variant AIMP2-DX2 RNA with a new transcript selectively exerting therapeutic activity in AIMP2-DX2-expressing lung cancer cells. The RNA replacement was employed via a high-fidelity trans-splicing reaction with the targeted residue of the AIMP2-DX2 transcript, but not with normal AIMP2 RNA, in the cells. Noticeably, the ribozyme could selectively deliver the activity of a suicide gene into the AIMP2-DX2 RNA expressing lung cancer cells and thereby specifically and effectively retard the growth of the cancer cells with prodrug treatment. Therefore, the AIMP2-DX2 RNA-targeting trans-splicing ribozyme could be a useful genetic agent for efficient therapy targeting lung cancer. [Copyright &y& Elsevier]

The full-length cytochrome c oxidase subunit I gene ( cox1) containing a group I intron was isolated from an important medical fungus Cordyceps militaris (Cordycipitaceae). The open reading frame (ORF) of 1,593 nucleotides encoded a predicted protein COX1 of 530 amino acids. The group I intron encoded a putative homing endonuclease (HE) with two LAGLIDADG motifs. RT-PCR and Northern analysis showed a mature transcript of spliced cox1. Both 5′exon-intron and intron-3′exon junctions were also found by RT-PCR, suggesting the possible presence of unspliced cox1 RNA in total RNA. Sequence comparison by BLASTn showed that the coding region of cox1 ( CRcox1) of C. militaris had significant similarities to those of related species (such as Cordyceps bassiana and C. brongniartii), while the intron had no significant homologous sequences of Cordycipitaceae fungi in NCBI database. The phylogenetic tree based on the CRcox1 confirmed the present taxonomic status of related species, but the cox1 introns were phylogenetically distinct. Compared to C. bassiana and C. brongniartii, the cox1 intron of C. militaris exhibited specific splicing site and different intronic ORF. The analysis of the folding RNA structures of the known cox1 introns from Cordyceps species showed different base pairs and conserved regions (P1-P10) in their structures. The present results provide useful information on the studies of cox1 intron splicing and Cordyceps evolution. [ABSTRACT FROM AUTHOR]

Recently, the number of cryptic species known has increased considerably, showing that species diversity has in many cases been underestimated in the past. Parmelia sulcata is a widely distributed species and one of the most common taxa in temperate Europe. The first intra-specific molecular studies on P. sulcata showed an unexpectedly high genetic variability. In the present work, we study the biodiversity of this taxon including specimens from four continents and using three molecular markers (nuITS, nuIGS rDNA, and partial β-tubulin gene). Two monophyletic groups of P. sulcata were encountered; one of these is epitypified as P. sulcata s. str and the other one is segregated as the new cryptic species P. encryptata sp. nov. Issues surrounding the lectotypification of Parmelia sulcata have also been elucidated. [ABSTRACT FROM AUTHOR]

Abstract: Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals. [Copyright &y& Elsevier]

In the leptosporangiate fern Osmunda regalis, cox1 gene is disrupted by a 1071-nucleotide-long group I intron that is homologous to the Marchantia polymorpha cox1 intron 4 (cox1i395g1). This intron, which shares 89% sequence identity with its bryophyte counterpart, lost the capacity to encode for a maturase due to insertion/deletion mutations. The cox1 coding region is interrupted by a stop codon in both exons. The cox1 transcript undergoes 58 C-to-U and 13 U-to-C conversions, including the suppression of two stop codons that result in the recovery of a functional cox1 ORF. Interestingly, 4 C-to-U conversions found in mRNA precursors showed that the O. regalis cox1i395g1 intron is efficiently edited. These modifications improved the sequence identity with the Marchantia cox1i395 intron. In particular, the RNA editing events affect regions involved in secondary and tertiary structures of the intron, restoring three base pairing in the structural P5a and P9 helices, and correcting a highly conserved U in the P7 helix that contributes to the catalytic core. Moreover, cox1 intron orthologous from three different fern species were found to be edited by both C-to-U and U-to-C conversions in P7 and P9. Thus, RNA editing helps to correct the conserved domains of group I introns in 'true ferns', suggesting a possible link between editing and splicing. We present here the first experimental evidence of RNA editing concerning a group I intron in plant organelles. [ABSTRACT FROM AUTHOR]

Since its initial description more than two decades ago, the ribosome bypass (or "hop") sequence of phage T4 stands out as a uniquely extreme example of programmed translational frameshifting. The gene for a DNA topoisomerase subunit of T4 has been split by a 1-kb insertion into two genes that retain topoisomerase function. A second 50-nt insertion, beginning with an in-phase stop codon, is inserted near the start of the newly created downstream gene 60. Instead of terminating at this stop codon, approximately half of the ribosomes skip 50 nucleotides and continue translation in a new reading frame. However, no functions, regulatory or otherwise, have been imputed for the truncated peptide that results from termination at codon 46 or for the bypass sequence itself. Moreover, how this unusual mRNA organization arose and why it is maintained have never been explained. We show here that a homing endonuclease (MobA) is encoded in the insertion that created gene 60, and the mobA gene together with the bypass sequence constitute a mobile DNA cassette. The bypass sequence provides protection against self-cleavage by the nuclease, whereas the nuclease promotes horizontal spread of the entire cassette to related bacteriophages. Group I introns frequently provide protection against self-cleavage by associated homing endonucleases. We present a scenario by which the bypass sequence, which is otherwise a unique genetic element, might have been derived from a degenerate group I intron. [ABSTRACT FROM AUTHOR]