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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]

Circular RNA (circRNA) has various advantages over linear mRNA that is gaining success as a new vaccine and therapeutic agent. Thus, circRNA and its engineering methods have attracted attention recently. In this study, we developed a new in vitro circRNA engineering method by end-to-end self-targeting and splicing (STS) reaction using Tetrahymena group I intron ribozyme. We found that only the P1 helix structure of the group I intron was enough to generate circRNA by STS reaction. The efficacy of circRNA generation by STS reaction was comparable to the method using a permuted intron-exon (PIE) reaction. However, an end-to-end STS reaction does not introduce any extraneous fragments, such as an intronic scar that can be generated by PIE reaction and might trigger unwanted innate immune responses in cells, into circRNA sequences. Moreover, generated circRNA was efficiently purified by ion pair-reversed phase high-pressure liquid chromatography and used for cell-based analysis. Of note, efficient protein expression and stability with least innate immune induction by the circRNA with coxsackievirus B3 IRES were observed in cells. In conclusion, our new in vitro circRNA strategy can effectively generate highly useful circRNAs in vitro as an alternative circRNA engineering method.

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]

Exserohilum turcicum and E. rostratum, two closely related fungal species, are both economically important pathogens but have quite different target hosts (specific to plants and cross-kingdom infection, respectively). In the present study, complete circular mitochondrial genomes of the two Exserohilum species were sequenced and de novo assembled, which mainly comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs, and a certain number of tRNAs and unidentified open reading frames (ORFs). Comparative analyses indicated that these two fungi had significant mitogenomic collinearity and consistent mitochondrial gene arrangement, yet with vastly different mitogenome sizes, 264,948 bp and 64,620 bp, respectively. By contrast with the 17 introns containing 17 intronic ORFs (one-to-one) in the E. rostratum mitogenome, E. turcicum involved far more introns (70) and intronic ORFs (126), which was considered as the main contributing factors of their mitogenome expansion/contraction. Within the generally intron-rich gene cox1, a total of 18 and 10 intron position classes (Pcls) were identified separately in the two mitogenomes. Moreover, 16.16% and 10.85% ratios of intra-mitogenomic repetitive regions were detected in E. turcicum and E. rostratum, respectively. Based on the combined mitochondrial gene dataset, we established a well-supported topology of phylogeny tree of 98 ascomycetes, implying that mitogenomes may act as an effective molecular marker for fungal phylogenetic reconstruction. Our results served as the first report on mitogenomes in the genus Exserohilum, and would have significant implications in understanding the origin, evolution and pathogenic mechanisms of this fungal lineage.

Crystallography is a major technique for determining large RNA structures. Obtaining diffraction-quality crystals has been the bottleneck. Although several RNA crystallization methods have been developed, the field strongly needs additional approaches. Here we invented an in crystallo selection strategy for identifying mutations that enhance a target RNA's crystallizability. The strategy includes constructing an RNA pool containing random mutations, obtaining crystals, and amplifying the sequences enriched by crystallization. We demonstrated a proof-of-principle application to the P4-P6 domain from the Tetrahymena ribozyme. We further determined the structures of four selected mutants. All four establish new crystal lattice contacts while maintaining the native structure. Three mutants achieve this by relocating bulges and one by making a helix more flexible. In crystallo selection provides opportunities to improve crystals of RNAs or RNA-ligand complexes. Our results also suggest that mutants may be rationally designed for crystallization by "walking" a bulge along the RNA chain.

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]

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.

Corynespora cassiicola, the causal agent of an extensive range of plant diseases worldwide, is a momentous fungus with diverse lifestyles and rich in intraspecies variations. In the present study, a total of 56 mitochondrial genomes of C. cassiicola were assembled (except two available online) and analyzed, of which 16 mitogenomes were newly sequenced here. All these circular mitochondrial DNA (mtDNA) molecules, ranging from 39,223 bp to 45,786 bp in length, comprised the same set of 13 core protein-coding genes (PCGs), two rRNAs and 27 tRNAs arranged in identical order. Across the above conserved genes, nad3 had the largest genetic distance between different isolates and was possibly subjected to positive selection pressure. Comparative mitogenomic analysis indicated that seven group I (IB, IC1, and IC2) introns with a length range of 1013–1876 bp were differentially inserted in three core PCGs (cox1, nad1, and nad5), resulting in the varied mitogenome sizes among C. cassiicola isolates. In combination with dynamic distribution of the introns, a well-supported mitogenome-wide phylogeny of the 56 C. cassiicola isolates revealed eight phylogenetic groups, which only had weak correlations with host range and toxin class. Different groups of isolates exhibited obvious differences in length and GC content of some genes, while a degree of variance in codon usage and tRNA structure was also observed. This research served as the first report on mitogenomic comparisons within C. cassiicola, and could provide new insights into its intraspecific microevolution and genetic diversity.

Orbilia xanthostigma, with golden yellow to yellow-orange apothecia and O. leucostigma, with white to very pale rose-lilaceous apothecia, were described by E.M. Fries over 200 years ago. Each of the two taxa, which are not easy to interpret because type material is lacking, was proposed in the past as lectotype of the genus Orbilia. In the here presented circumscription, which follows the current usage, O. xanthostigma is among the most frequently recorded species of the genus, whereas O. leucostigma appears to be much less common. Both grow gregariously on decorticated hygric gymno- and angiosperm wood or rarely bark and show a worldwide distribution. They are characterised by minute, strongly curved, warted ascospores and a dicranidion-like anamorph. Except for apothecial colour, there are no other notable morphological differences between them, either in the teleomorph (asci, ascospores, paraphyses) or anamorph (conidiophores, conidia). Because of their strong similarities, the two taxa have often been treated as infraspecific variants (subspecies, varieties) of a single species or even as synonyms. In order to overcome the ambiguities associated with the two names, O. delicatula, a name proposed by P.A. Karsten for a collection with golden yellow apothecia, was suggested by B. Spooner as a replacement name for them. The present study reveals unexpectedly high ITS and LSU variation within a morphologically extremely homogeneous group, representing over 16 more or less invariable genotypes whenever more than one sample with a sequence was available. ITS and LSU rDNA data from European (Luxembourg, Germany, Ukraine) and Macaronesian (Tenerife) collections suggest that the two colour variants represent two distinct species with a 16.5% ITS and 3.5% LSU D1–D2 distance, but very low infraspecific variation (0.2% ITS, 0% LSU). A sample with scattered yellow apothecia from Luxembourg on a xeric branch deviates from typical European-Macaronesian O. xanthostigma by 4% (ITS) and 0.7% (LSU). Further available sequences from samples from Asia, New Zealand and North America with mainly yellow apothecia clustered in various other clades that represent further distinct genotypes. In the absence of morphological characteristics, none of these genotypes are given names pending further investigation. Only two of these genotypes are sufficiently distinct to be recognised morphologically: O. aureocrenulata from tropical, Middle and South America, with golden yellow apothecia with a crenulate margin and prominent stipe, and O. xanthoflexa from temperate, northeastern North America with yellow sessile apothecia with a smooth margin and larger, less curved, smooth-walled ascospores. [ABSTRACT FROM AUTHOR]

Abstract 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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (2024, by The Mycological Society of Japan.)

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]

Group I introns are ribozymes (catalytic RNAs) that excise themselves from RNA primary transcripts by catalyzing two successive transesterification reactions. These cis-splicing ribozymes can be converted into trans-splicing ribozymes, which can modify the sequence of a separate substrate RNA, both in vitro and in vivo. Previous work on trans-splicing ribozymes has mostly focused on the 16S rRNA group I intron ribozyme from Tetrahymena thermophila. Here, we test the trans-splicing potential of the tRNA(Ile) group I intron ribozyme from the bacterium Azoarcus. This ribozyme is only half the size of the Tetrahymena ribozyme and folds faster into its active conformation in vitro. Our results showed that in vitro, the Azoarcus and Tetrahymena ribozymes favored the same set of splice sites on a substrate RNA. Both ribozymes showed the same trans-splicing efficiency when containing their individually optimized 5' terminus. In contrast to the previously optimized 5'-terminal design of the Tetrahymena ribozyme, the Azoarcus ribozyme was most efficient with a trans-splicing design that resembled the secondary structure context of the natural cis-splicing Azoarcus ribozyme, which includes base-pairing between the substrate 5' portion and the ribozyme 3' exon. These results suggested preferred trans-splicing interactions for the Azoarcus ribozyme under near-physiological in vitro conditions. Despite the high activity in vitro, however, the splicing efficiency of the Azoarcus ribozyme in Escherichia coli cells was significantly below that of the Tetrahymena ribozyme.

Abstract Background There are six known families of homing endonucleases, LAGLIDADG, GIY-YIG, HNH, His-Cys box, PD-(D/E)-XK, and EDxHD, which are characterized by their conserved residues. Previously, we discovered a novel homing endonuclease F-CphI encoded by ORF177 of cyanophage S-PM2. F-CphI does not resemble any characterized homing endonucleases. Instead, the C-terminus of F-CphI aligns well with the N-terminal catalytic domain of a Holliday junction DNA resolvase, phage T4 endonuclease VII (Endo VII). Results A PSI-BLAST search resulted in a total of 313 Endo VII motif–containing sequences in sequenced genomes. Multiple sequence alignment showed that the catalytically important residues of T4 Endo VII were all well conserved in these proteins. Our site-directed mutagenesis studies further confirmed that the catalytically important residues of T4 Endo VII were also essential for F-CphI activity, and thus F-CphI might use a similar protein fold as Endo VII for DNA cleavage. A phylogenetic tree of the Endo VII motif–containing sequences showed that putative resolvases grouped into one clade while putative homing endonucleases and restriction endonucleases grouped into another clade. Conclusions Based on the unique conserved residues, we proposed that F-CphI represents a new homing endonuclease family, which was named the DHHRN family. Our phylogenetic analysis could be used to predict the functions of many previously unknown proteins.

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]

Group I catalytic introns are widespread in bacterial, archaeal, viral, organellar, and some eukaryotic genomes, where they are reported to provide regulatory functions. The group I introns are currently divided into five types (A-E), which are themselves distributed into several subtypes, with the exception of group I type D intron (GI-D). GI-D introns belong to the rarest group with only 17 described to date, including only one with a putative role reported in fungi, where it would interfere with an adaptive response in the cytochrome b (COB) gene to quinone outside inhibitor (QoI) fungicide resistance. Using homology search methods taking into account both conserved sequences and RNA secondary structures, we analysed the mitochondrial genomes or COB genes of 169 fungal species, including some frequently under QoI selection pressure. These analyses have led to the identification of 216 novel GI-D introns, and the definition of three distinct subtypes, one of which being linked with a functional activity. We have further uncovered a homing site for this GI-D intron type, which helps refine the accepted model of quinone outside inhibitor resistance, whereby mobility of the intron across fungal mitochondrial genomes, would influence a fungus ability to develop resistance to QoIs. [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]

Introns are ubiquitous in eukaryotic genomes and have long been considered as ‘junk RNA’ but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

A hallmark of sea anemone mitochondrial genomes (mitogenomes) is the presence of complex catalytic group I introns. Here, we report the complete mitogenome and corresponding transcriptome of the carpet sea anemone Stichodactyla haddoni (family Stichodactylidae). The mitogenome is vertebrate-like in size, organization, and gene content. Two mitochondrial genes encoding NADH dehydrogenase subunit 5 (ND5) and cytochrome c oxidase subunit I (COI) are interrupted with complex group I introns, and one of the introns (ND5-717) harbors two conventional mitochondrial genes (ND1 and ND3) within its sequence. All the mitochondrial genes, including the group I introns, are expressed at the RNA level. Nonconventional and optional mitochondrial genes are present in the mitogenome of S. haddoni. One of these gene codes for a COI-884 intron homing endonuclease and is organized in-frame with the upstream COI exon. The insertion-like orfA is expressed as RNA and translocated in the mitogenome as compared with other sea anemones. Phylogenetic analyses based on complete nucleotide and derived protein sequences indicate that S. haddoni is embedded within the family Actiniidae, a finding that challenges current taxonomy.

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]

Abstract Nuclear group I introns are parasitic mobile genetic elements occurring in the ribosomal RNA genes of a large variety of microbial eukaryotes. In Acanthamoeba , group I introns were found occurring in the 18S rDNA at four distinct insertion sites. Introns are present as single elements in various strains belonging to four genotypes, T3 (A. griffini), T4 (A. castellanii complex), T5 (A. lenticulata) and T15 (A. jacobsi). While multiple introns can frequently be found in the rDNA of several algae, fungi and slime moulds, they are usually rare and present as single elements in amoebae. We reported herein the characterization of an A. lenticulata strain containing two introns in its 18S rDNA. They are located to already known sites and show basal relationships with respective homologous introns present in the other T5 strains. This is the first and unique reported case of multiple nuclear introns in Acanthamoeba. [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]

The intron located between the first and second nucleotide of the leucine tRNA anticodon is the only representative of group I introns in higher plants. In this paper, for the first time the intron sequence of the plastid trnL gene is described in 16 legume species, and putative secondary structures of the entire intron and some of its functional domains are reconstructed. It has been found that genera of the Fabaceae family, as well as species within a single genus, are highly diverse in this sequence. Single nucleotide polymorphisms have been found in sequences of the catalytic center, believed to be highly conserved.

Circular RNA (circRNA) has various advantages over linear mRNA that is gaining success as a new vaccine and therapeutic agent. Thus, circRNA and its engineering methods have attracted attention recently. In this study, we developed a new in vitro circRNA engineering method by end-to-end self-targeting and splicing (STS) reaction using Tetrahymena group I intron ribozyme. We found that only the P1 helix structure of the group I intron was enough to generate circRNA by STS reaction. The efficacy of circRNA generation by STS reaction was comparable to the method using a permuted intron-exon (PIE) reaction. However, an end-to-end STS reaction does not introduce any extraneous fragments, such as an intronic scar that can be generated by PIE reaction and might trigger unwanted innate immune responses in cells, into circRNA sequences. Moreover, generated circRNA was efficiently purified by ion pair-reversed phase high-pressure liquid chromatography and used for cell-based analysis. Of note, efficient protein expression and stability with least innate immune induction by the circRNA with coxsackievirus B3 IRES were observed in cells. In conclusion, our new in vitro circRNA strategy can effectively generate highly useful circRNAs in vitro as an alternative circRNA engineering method., Competing Interests: K.H.L., S.K., J.S., S.R.H., and J.H.K. are employee of Rznomics Inc. S.-W.L. is CEO of Rznomics Inc., (© 2023 The Author(s).)

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]

Abstract Various strains belonging to three Acanthamoeba species, A. griffini (genotype T3), A. lenticulata (T5), and A. jacobsi (T15), have group I introns in their 18S rRNA genes. Group I introns are self-splicing ribozymes that can spread among host lineages either through an intron-encoded endonuclease at the DNA level, or by reverse splicing during the RNA cycle. In Acanthamoeba , introns belong to the subclass IC1, they are located at one out four positions within the rRNA, show low identity values and all lack open reading frames to encode for an endonuclease. Uncharacterized introns from strains of another genotype, T4 (A. castellanii complex), resemble those of genotype T3, and at least one of them contains a non-functional endonuclease gene. Here, we analyzed all available data on Acanthamoeba 18S rDNA sequences to identify the possible presence of open reading frames that could encode endonucleases. We found a total of eight 18S rDNA sequences, all from T4 strains, that have introns containing putative non-functional endonuclease genes. Furthermore, two distinct endonucleases can be identified that are differently inserted in unrelated introns. [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]