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The Ophiostomatales are of economic concern, as many are blue-stain fungi and some are plant pathogens. The mitogenomes of members assigned to this order exhibit size polymorphism despite having highly conserved gene order, owing to the variable number of introns and intron insertion sites. In this work, eleven blue-stain fungi, including nine strains of Ophiostoma ips with a varied distribution across North America and New Zealand, were sequenced and compared with other members of the Ophiostomatales . A pan-mitogenome intron landscape has been prepared to demonstrate the distribution of the mobile genetic elements and to provide insight into the evolutionary dynamics of introns among members of this group of fungi. The size variation among these mitogenomes (from about 23.8 kb to 152 kb) shows high correlation to the presence and absence of introns. Examples of complex or nested introns composed of two or three intron modules have been observed in some O. ips strains. RNA-seq data suggests possible splicing pathways with regard to resolving these complex introns. Mitochondrial DNA and RNA data for O. ips provides the basis for future studies relating to gene annotation, alternative splicing, evolutionary intron dynamics, and taxonomic investigations for members of the Ophiostomatales .

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.

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.

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.

Group II introns are large catalytic RNAs (ribozymes) which are found in bacteria and organellar genomes of several lower eukaryotes, but are particularly prevalent within the mitochondrial genomes (mtDNA) in plants, where they reside in numerous critical genes. Their excision is therefore essential for mitochondria biogenesis and respiratory functions, and is facilitated in vivo by various protein cofactors. Typical group II introns are classified as mobile genetic elements, consisting of the self-splicing ribozyme and its intron-encoded maturase protein. A hallmark of maturases is that they are intron specific, acting as cofactors which bind their own cognate containing pre-mRNAs to facilitate splicing. However, the plant organellar introns have diverged considerably from their bacterial ancestors, such as they lack many regions which are necessary for splicing and also lost their evolutionary related maturase ORFs. In fact, only a single maturase has been retained in the mtDNA of various angiosperms: the matR gene encoded in the fourth intron of the NADH-dehydrogenase subunit 1 (nad1 intron 4). Their degeneracy and the absence of cognate ORFs suggest that the splicing of plant mitochondria introns is assisted by trans-acting cofactors. Interestingly, in addition to MatR, the nuclear genomes of angiosperms also harbor four genes (nMat 1-4), which are closely related to maturases and contain N-terminal mitochondrial localization signals. Recently, we established the roles of two of these paralogs in Arabidopsis, nMAT1 and nMAT2, in the splicing of mitochondrial introns. In addition to the nMATs, genetic screens led to the identification of other genes encoding various factors, which are required for the splicing and processing of mitochondrial introns in plants. In this review we will summarize recent data on the splicing and processing of mitochondrial introns and their implication in plant development and physiology, with a focus on maturases and their accessory splicing cofactors.

Group II introns are ribozymes that can excise themselves from precursor-RNA transcripts, but plant organellar group II introns have structural deviations that inhibit ribozyme activity. Therefore, splicing of these introns requires the assistance of nuclear- and/or organellar-encoded splicing factors; however, how these splicing factors function remains unclear. In this study, we report the functions and interactions of two splicing factors, PPR-SMR1 and Zm-mCSF1, in intron splicing in maize mitochondria. PPR-SMR1 is a SMR domain-containing pentatricopeptide repeat (PPR) protein and Zm-mCSF1 is a CRM domain-containing protein, and both are targeted to mitochondria. Loss-of-function mutations in each of them severely arrests embryogenesis and endosperm development in maize. Functional analyses indicate that PPR-SMR1 and Zm-mCSF1 are required for the splicing of most mitochondrial group II introns. Among them, nad2-intron 2 and 3, and nad5-intron 1 are PPR-SMR1/Zm-mCSF1-dependent introns. Protein interaction assays suggest that PPR-SMR1 can interact with Zm-mCSF1 through its N-terminus, and that Zm-mCSF1 is self-interacting. Our findings suggest that PPR-SMR1, a novel splicing factor, acts in the splicing of multiple group II introns in maize mitochondria, and the protein–protein interaction between it and Zm-mCSF1 might allow the formation of large macromolecular splicing complexes.

ABSTRACT Reverse transcriptases (RTs) catalyze the polymerization of DNA from an RNA template. These enzymes were first discovered in RNA tumor viruses in 1970, but it was not until 1989 that they were found in prokaryotes as a key component of retrons. Apart from RTs encoded by the ‘selfish’ mobile retroelements known as group II introns, prokaryotic RTs are extraordinarily diverse, but their function has remained elusive. However, recent studies have revealed that different lineages of prokaryotic RTs, including retrons, those associated with CRISPR-Cas systems, Abi-like RTs and other yet uncharacterized RTs, are key components of different lines of defense against phages and other mobile genetic elements. Prokaryotic RTs participate in various antiviral strategies, including abortive infection (Abi), in which the infected cell is induced to commit suicide to protect the host population, adaptive immunity, in which a memory of previous infection is used to build an efficient defense, and other as yet unidentified mechanisms. These prokaryotic enzymes are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, we discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems. Prokaryotic reverse transcriptases (RTs) are attracting considerable attention, both for use in cutting-edge technologies, such as genome editing, and as an emerging research topic. In this review, the authors discuss what is known about prokaryotic RTs, and the exciting evidence for their domestication from retroelements to create specialized defense systems.

Group II introns are catalytic RNAs that combine self-splicing ribozyme activity with mobility and have played major roles in shaping organellar genome evolution. In green macroalgae of the genus Ulva, organellar genomes are highly compact, yet they harbor unusually diverse and dynamic repertoires of group II introns. To understand how organellar group II introns diversify and persist within compact organellar genomes, we performed a comparative analysis of mitochondrial and chloroplast group II introns across Ulva, integrating secondary structure reconstruction, intron occurrence patterns, and phylogenetic inference based on both conserved intron RNA regions and intron-encoded proteins (IEPs), including reverse transcriptase/maturase (RT/M) and LAGLIDADG homing endonuclease (LHE). A total of 168 mitochondrial and 123 chloroplast introns were identified and classified into 32 families belonging to seven major subgroups (IIA1-RT/M, IIA2-RT/M, IIB1-RT/M, IIB1-LHE, IIB2-RT/M, IIB2-LHE, and IIB-like). Most intron families retain the canonical six-domain architecture (DI–DVI), but four mitochondrial IIA families display a seven-domain configuration generated by the lineage-specific insertion of an additional stem-loop structure (DIIIa). Phylogenetic analyses revealed a high degree of congruence, supporting persistent coevolution between RNA scaffolds and their IEPs. Notably, the LHE-encoding families were scattered across distinct IIB lineages instead of forming a single clade, suggesting that at least two independent invasion events occurred within the IIB1 and IIB2 lineages. Analysis of intron occurrence frequency revealed an evolutionary continuum ranging from structurally intact and broadly distributed families to lineage-specific families exhibiting progressive scaffold degeneration, with the chloroplast infA-62 family representing a stably inherited lineage maintained through vertical transmission. These results suggest that organellar group II introns in Ulva evolve through coordinated scaffold remodeling, enzymatic replacement, and differential distribution patterns across genomic compartments, highlighting Ulva organellar genomes as a valuable comparative model for investigating the long-term evolution of mobile ribozymes within compact genomic environments.

Introns were traditionally thought to be rare in bacteria, yet their occurrence and diversity may have been underestimated. Here, we present the first comprehensive overview of group I and group II introns in Patescibacteria. While most introns are readily identified, group I introns inserted at position 35/36 within the anticodon loop often escape detection by standard annotation tools; through experimental verification, we demonstrate that these introns are accurately spliced despite their unusual insertion site. Notably, approximately 40% of genomes in both Patescibacteria and Cyanobacteriota harbor group I introns; however, while around 20% of Cyanobacteriota genomes also contain group II introns, none were detected in Patescibacteria. These results illustrate a previously overlooked phylogenetic distribution of group I and group II introns across the bacterial domain.

Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCR-amplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon , Cenchrus , Solanum , and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus , Cynodon , Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species’ introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified.