Intron Mobility Research Articles

Digital PCR: a tool in clostridial mutant selection and detection.

The ClosTron mutagenesis system has enabled researchers to efficiently edit the clostridial genome. Since site-specific insertion of the mobile ClosTron insert may cause errors, validation is key. In this paper we describe the use of digital PCR (dPCR) as an alternative tool in selecting clostridial mutant strains. Clostridium perfringens chitinase mutant strains were constructed in which the mobile ClosTron intron was inserted into one of the chitinase genes. On-target insertion of the mobile intron was validated through conventional PCR. In order to confirm the absence of off-target insertions, dPCR was used to determine the amount of the ClosTron intron as well as the amount of a reference gene, located in close proximity to the interrupted gene. Subsequently, mutant strains containing an equivalent amount of both genes were selected as these do not contain additional off-target mobile ClosTron inserts. The outcome of this selection procedure was confirmed through a validated PCR-based approach. In addition to its application in mutant selection, dPCR can be used in other aspects of clostridial research, such as the distinction and easy quantification of different types of strains (wildtype vs. mutant) in complex matrices, such as faecal samples, a process in which other techniques are hampered by bacterial overgrowth (plating) or inhibition by matrix contaminants (qPCR). This research demonstrates that dPCR is indeed a high-throughput method in the selection of clostridial insertion mutants as well as a robust and accurate tool in distinguishing between wildtype and mutant C. perfringens strains, even in a complex matrix such as faeces. KEY POINTS: • Digital PCR as an alternative in ClosTron mutant selection • Digital PCR is an accurate tool in bacterial quantification in a complex matrix • Digital PCR is an alternative tool with great potential to microbiological research.

Categorizing 161 plant (streptophyte) mitochondrial group II introns into 29 families of related paralogues finds only limited links between intron mobility and intron-borne maturases

Group II introns are common in the two endosymbiotic organelle genomes of the plant lineage. Chloroplasts harbor 22 positionally conserved group II introns whereas their occurrence in land plant (embryophyte) mitogenomes is highly variable and specific for the seven major clades: liverworts, mosses, hornworts, lycophytes, ferns, gymnosperms and flowering plants. Each plant group features “signature selections” of ca. 20–30 paralogues from a superset of altogether 105 group II introns meantime identified in embryophyte mtDNAs, suggesting massive intron gains and losses along the backbone of plant phylogeny. We report on systematically categorizing plant mitochondrial group II introns into “families”, comprising evidently related paralogues at different insertion sites, which may even be more similar than their respective orthologues in phylogenetically distant taxa. Including streptophyte (charophyte) algae extends our sampling to 161 and we sort 104 streptophyte mitochondrial group II introns into 25 core families of related paralogues evidently arising from retrotransposition events. Adding to discoveries of only recently created intron paralogues, hypermobile introns and twintrons, our survey led to further discoveries including previously overlooked “fossil” introns in spacer regions or e.g., in the rps8 pseudogene of lycophytes. Initially excluding intron-borne maturase sequences for family categorization, we added an independent analysis of maturase phylogenies and find a surprising incongruence between intron mobility and the presence of intron-borne maturases. Intriguingly, however, we find that several examples of nuclear splicing factors meantime characterized simultaneously facilitate splicing of independent paralogues now placed into the same intron families. Altogether this suggests that plant group II intron mobility, in contrast to their bacterial counterparts, is not intimately linked to intron-encoded maturases.

Structures of a mobile intron retroelement poised to attack its structured DNA target.

Group II introns are ribozymes that catalyze their self-excision and function as retroelements that invade DNA. As retrotransposons, group II introns form ribonucleoprotein (RNP) complexes that roam the genome, integrating by reversal of forward splicing. Here we show that retrotransposition is achieved by a tertiary complex between a structurally elaborate ribozyme, its protein mobility factor, and a structured DNA substrate. We solved cryo-electron microscopy structures of an intact group IIC intron-maturase retroelement that was poised for integration into a DNA stem-loop motif. By visualizing the RNP before and after DNA targeting, we show that it is primed for attack and fits perfectly with its DNA target. This study reveals design principles of a prototypical retroelement and reinforces the hypothesis that group II introns are ancient elements of genetic diversification.

Phylogenetic divergences in brown rot fungal pathogens of Monilinia species from a worldwide collection: inferences based on the nuclear versus mitochondrial genes

BackgroundPhylogenetic analyses for plant pathogenic fungi explore many questions on diversities, relationships, origins, and divergences of populations from different sources such as species, host, and geography. This information is highly valuable, especially from a large global sampling, to understand the evolutionary paths of the pathogens worldwide. Monilinia fructicola and M. laxa are two important fungal pathogens of stone fruits that cause the widespread disease commonly known as brown rot. Three nuclear genes (Calmodulin, SDHA, TEF1α) and three mitochondrial genes (Cytochrome_b, NAD2, and NAD5) of the two pathogen species from a worldwide collection including five different countries from four different continents were studied in this work.ResultsBoth Maximum Likelihood and Bayesian approaches were applied to the data sets, and in addition, Maximum Parsimony based approaches were used for the regions having indel polymorphisms. Calmodulin, SDHA, NAD2, and NAD5 regions were found phylogenetically informative and utilized for phylogenetics of Monilinia species for the first time. Each gene region presented a set of haplotypes except Cytochrome_b, which was monomorphic. According to this large collection of two Monilinia species around the world, M. fructicola showed more diversity than M. laxa, a result that should be carefully considered, as M. fructicola is known to be a quarantine pathogen. Moreover, the other two mitochondrial genes (NAD2 and NAD5) did not have any substitution type mutations but presented an intron indel polymorphism indicating the contribution of introns as well as mobile introns to the fungal diversity and evolution. Based on the concatenated gene sets, nuclear DNA carries higher mutations and uncovers more phylogenetic clusters in comparison to the mitochondrial DNA-based data for these fungal species.ConclusionsThis study provides the most comprehensive knowledge on the phylogenetics of both nuclear and mitochondrial genes of two prominent brown rot pathogens, M. fructicola and M. laxa. Based on the regions used in this study, the nuclear genes resolved phylogenetic branching better than the mitochondrial genes and discovered new phylogenetic lineages for these species.

Mobile group I introns at nuclear rDNA position L2066 harbor sense and antisense homing endonuclease genes intervened by spliceosomal introns

BackgroundMobile group I introns encode homing endonucleases that confer intron mobility initiated by a double-strand break in the intron-lacking allele at the site of insertion. Nuclear ribosomal DNA of some fungi and protists contain mobile group I introns harboring His-Cys homing endonuclease genes (HEGs). An intriguing question is how protein-coding genes embedded in nuclear ribosomal DNA become expressed. To address this gap of knowledge we analyzed nuclear L2066 group I introns from myxomycetes and ascomycetes.ResultsA total of 34 introns were investigated, including two identified mobile-type introns in myxomycetes with HEGs oriented in sense or antisense directions. Intriguingly, both HEGs are interrupted by spliceosomal introns. The intron in Didymium squamulosum, which harbors an antisense oriented HEG, was investigated in more detail. The group I intron RNA self-splices in vitro, thus generating ligated exons and full-length intron circles. The intron HEG is expressed in vivo in Didymium cells, which involves removal of a 47-nt spliceosomal intron (I-47) and 3′ polyadenylation of the mRNA. The D. squamulosum HEG (lacking the I-47 intron) was over-expressed in E. coli, and the corresponding protein was purified and shown to confer endonuclease activity. The homing endonuclease was shown to cleave an intron-lacking DNA and to produce a pentanucleotide 3′ overhang at the intron insertion site.ConclusionsThe L2066 family of nuclear group I introns all belong to the group IE subclass. The D. squamulosum L2066 intron contains major hallmarks of a true mobile group I intron by encoding a His-Cys homing endonuclease that generates a double-strand break at the DNA insertion site. We propose a potential model to explain how an antisense HEG becomes expressed from a nuclear ribosomal DNA locus.

Mitogenomics and mitochondrial gene phylogeny decipher the evolution of Saccharomycotina yeasts.

Saccharomycotina yeasts belong to diverse clades within the kingdom of fungi and are important to human everyday life. This work investigates the evolutionary relationships among these yeasts from a mitochondrial (mt) genomic perspective. A comparative study of 155 yeast mt genomes representing all major phylogenetic lineages of Saccharomycotina was performed, including genome size and content variability, intron and intergenic regions’ diversity, genetic code alterations, and syntenic variation. Findings from this study suggest that mt genome size diversity is the result of a ceaseless random process, mainly based on genetic recombination and intron mobility. Gene order analysis revealed conserved syntenic units and many occurring rearrangements, which can be correlated with major evolutionary events as shown by the phylogenetic analysis of the concatenated mt protein matrix. For the first time, molecular dating indicated a slower mt genome divergence rate in the early stages of yeast evolution, in contrast with a faster rate in the late evolutionary stages, compared to their nuclear time divergence. Genetic code reassignments of mt genomes are a perpetual process happening in many different parallel evolutionary steps throughout the evolution of Saccharomycotina. Overall, this work shows that phylogenetic studies based on the mt genome of yeasts highlight major evolutionary events.

Identification of Group II Intron RmInt1 Binding Sites in a Bacterial Genome.

RmInt1 is a group II intron encoding a reverse transcriptase protein (IEP) lacking the C-terminal endonuclease domain. RmInt1 is an efficient mobile retroelement that predominantly reverse splices into the transient single-stranded DNA at the template for lagging strand DNA synthesis during host replication, a process facilitated by the interaction of the RmInt1 IEP with DnaN at the replication fork. It has been suggested that group II intron ribonucleoprotein particles bind DNA nonspecifically, and then scan for their correct target site. In this study, we investigated RmInt1 binding sites throughout the Sinorhizobium meliloti genome, by chromatin-immunoprecipitation coupled with next-generation sequencing. We found that RmInt1 binding sites cluster around the bidirectional replication origin of each of the three replicons comprising the S. meliloti genome. Our results provide new evidence linking group II intron mobility to host DNA replication.

A new RNA-DNA interaction required for integration of group II intron retrotransposons into DNA targets.

Mobile group II introns are site-specific retrotransposable elements abundant in bacterial and organellar genomes. They are composed of a large and highly structured ribozyme and an intron-encoded reverse transcriptase that binds tightly to its intron to yield a ribonucleoprotein (RNP) particle. During the first stage of the mobility pathway, the intron RNA catalyses its own insertion directly into the DNA target site. Recognition of the proper target rests primarily on multiple base-pairing interactions between the intron RNA and the target DNA, while the protein makes contacts with only a few target positions by yet-unidentified mechanisms. Using a combination of comparative sequence analyses and in vivo mobility assays we demonstrate the existence of a new base-pairing interaction named EBS2a–IBS2a between the intron RNA and its DNA target site. This pairing adopts a Watson–Crick geometry and is essential for intron mobility, most probably by driving unwinding of the DNA duplex. Importantly, formation of EBS2a–IBS2a also requires the reverse transcriptase enzyme which stabilizes the pairing in a non-sequence-specific manner. In addition to bringing to light a new structural device that allows subgroup IIB1 and IIB2 introns to invade their targets with high efficiency and specificity our work has important implications for the biotechnological applications of group II introns in bacterial gene targeting.

Mutational Effects of Mobile Introns on the Mitochondrial Genomes of Metschnikowia Yeasts

It has been argued that DNA repair by homologous recombination in the context of endonuclease-mediated cleavage can cause mutations. To better understand this phenomenon, we examined homologous recombination following endonuclease cleavage in a native genomic context: the movement of self-splicing introns in the mitochondrial genomes of Metschnikowia yeasts. Self-splicing mitochondrial introns are mobile elements, which can copy and paste themselves at specific insertion sites in mitochondrial DNA using a homing endonuclease in conjunction with homologous recombination. Here, we explore the mutational effects of self-splicing introns by comparing sequence variation within the intron-rich cox1 and cob genes from 71 strains (belonging to 40 species) from the yeast genus Metschnikowia. We observed a higher density of single nucleotide polymorphisms around self-splicing-intron insertion sites. Given what is currently known about the movement of organelle introns, it is likely that their mutational effects result from the high binding affinity of endonucleases and their interference with repair machinery during homologous recombination (or, alternatively, via gene conversion occurring during the intron insertion process). These findings suggest that there are fitness costs to harbouring self-splicing, mobile introns and will help us better understand the risks associated with modern biotechnologies that use endonuclease-mediated homologous recombination, such as CRISPR-Cas9 gene editing.

Pan-Mitogenomics Approach Discovers Diversity and Dynamism in the Prominent Brown Rot Fungal Pathogens.

Monilinia fructicola and Monilinia laxa species are the most destructive and economically devastating fungal plant pathogens causing brown rot disease on stone and pome fruits worldwide. Mitochondrial genomes (mitogenomes) play critical roles influencing the mechanisms and directions of the evolution of fungal pathogens. The pan-mitogenomics approach predicts core and accessory regions of the mitochondrial genomes and explains the gain or loss of variation within and between species. The present study is a fungal pan-mitogenome of M. fructicola (N = 8) and M. laxa (N = 8) species. The completely sequenced and annotated mitogenomes showed high variability in size within and between the species. The mitogenomes of M. laxa were larger, ranging from 178,351 to 179,780bp, than the mitogenomes of M. fructicola, ranging from 158,607 to 167,838bp. However, size variation within the species showed that M. fructicola isolates were more variable in the size range than M. laxa isolates. All the mitogenomes included conserved mitochondrial genes, as well as variable regions including different mobile introns encoding homing endonucleases or maturase, non-coding introns, and repetitive elements. The linear model analysis supported the hypothesis that the mitogenome size expansion is due to presence of variable (accessory) regions. Gene synteny was mostly conserved among all samples, with the exception for order of the rps3 in the mitogenome of one isolate. The mitogenomes presented AT richness; however, A/T and G/C skew varied among the mitochondrial genes. The purifying selection was detected in almost all the protein-coding genes (PCGs) between the species. However, cytochrome b was the only gene showing a positive selection signal among the total samples. Combined datasets of amino acid sequences of 14 core mitochondrial PCGs and rps3 obtained from this study together with published mitochondrial genome sequences from some other species from Heliotales were used to infer a maximum likelihood (ML) phylogenetic tree. ML tree indicated that both Monilinia species highly diverged from each other as well as some other fungal species from the same order. Mitogenomes harbor much information about the evolution of fungal plant pathogens, which could be useful to predict pathogenic life strategies.

Insights into genomic evolution from the chromosomal and mitochondrial genomes of Ustilaginoidea virens

Ustilaginoidea virens, the causal agent of rice false smut, is an economically important filamentous fungal pathogen. A high-quality reference genome of U. virens promotes understanding of molecular mechanisms underlying its virulence and pathogenicity. Here, we report the first chromosome-level assembly of U. virens genome consisting of seven chromosomes ranging from 2.4 to 7.5 Mb. The assembly has dramatic improvements over previous assemblies, including considerably longer contigs, higher proportion of repetitive elements and more functionally annotated genes. Phylogenetic analyses revealed an extremely low intraspecific sequence divergence in U. virens. By contrast, intraspecific genome comparisons uncovered dynamic genomic alterations including massive structural variations and widespread lineage-specific regions (LSRs) among U. virens strains, which were mainly generated by recent burst of retrotransposons. Genomic plasticity created by structural variations and LSRs might drive rapid evolution of U. virens. High-quality mitochondrial genomes of eight U. virens strains exhibit size variations from 94 to 102 kb. Consistently, U. virens contains conserved lengths of exons and highly dynamic mobile introns, which contribute to intraspecific size variations due to gain/loss of homing endonuclease genes. This study highlights unique characteristics in nuclear and mitochondrial genomic divergence and provides new insights into genomic and mitochondrial evolution of U. virens.

The Mitochondrial Genome of a Plant Fungal Pathogen Pseudocercospora fijiensis (Mycosphaerellaceae), Comparative Analysis and Diversification Times of the Sigatoka Disease Complex Using Fossil Calibrated Phylogenies.

Mycosphaerellaceae is a highly diverse fungal family containing a variety of pathogens affecting many economically important crops. Mitochondria play a crucial role in fungal metabolism and in the study of fungal evolution. This study aims to: (i) describe the mitochondrial genome of Pseudocercospora fijiensis, and (ii) compare it with closely related species (Sphaerulina musiva, S. populicola, P. musae and P. eumusae) available online, paying particular attention to the Sigatoka disease’s complex causal agents. The mitochondrial genome of P. fijiensis is a circular molecule of 74,089 bp containing typical genes coding for the 14 proteins related to oxidative phosphorylation, 2 rRNA genes and a set of 38 tRNAs. P. fijiensis mitogenome has two truncated cox1 copies, and bicistronic transcription of nad2-nad3 and atp6-atp8 confirmed experimentally. Comparative analysis revealed high variability in size and gene order among selected Mycosphaerellaceae mitogenomes likely to be due to rearrangements caused by mobile intron invasion. Using fossil calibrated Bayesian phylogenies, we found later diversification times for Mycosphaerellaceae (66.6 MYA) and the Sigatoka disease complex causal agents, compared to previous strict molecular clock studies. An early divergent Pseudocercospora fijiensis split from the sister species P. musae + P. eumusae 13.31 MYA while their sister group, the sister species P. eumusae and P. musae, split from their shared common ancestor in the late Miocene 8.22 MYA. This newly dated phylogeny suggests that species belonging to the Sigatoka disease complex originated after wild relatives of domesticated bananas (section Eumusae; 27.9 MYA). During this time frame, mitochondrial genomes expanded significantly, possibly due to invasions of introns into different electron transport chain genes.

A New Type of Circular RNA derived from Nonconventional Introns in Nuclear Genes of Euglenids

Nuclear protein-coding genes of euglenids (Discoba, Euglenozoa, Euglenida) contain conventional (spliceosomal) and nonconventional introns. The latter have been found only in euglenozoans. A unique feature of nonconventional introns is the ability to form a stable and slightly conserved RNA secondary structure bringing together intron ends and placing adjacent exons in proximity. To date, little is known about the mechanism of their excision (e.g. whether it involves the spliceosome or not). The tubA gene of Euglena gracilis harbors three conventional and three nonconventional introns. While the conventional introns are excised as lariats, nonconventional introns are present in the cell solely as circular RNAs with full-length ends. Based on this discovery as well as on previous observations indicating that nonconventional introns are observed frequently at unique positions of genes, we suggest that this new type of intronic circRNA might play a role in intron mobility.

Siberian larch (Larix sibirica Ledeb.) mitochondrial genome assembled using both short and long nucleotide sequence reads is currently the largest known mitogenome

BackgroundPlant mitochondrial genomes (mitogenomes) can be structurally complex while their size can vary from ~ 222 Kbp in Brassica napus to 11.3 Mbp in Silene conica. To date, in comparison with the number of plant species, only a few plant mitogenomes have been sequenced and released, particularly for conifers (the Pinaceae family). Conifers cover an ancient group of land plants that includes about 600 species, and which are of great ecological and economical value. Among them, Siberian larch (Larix sibirica Ledeb.) represents one of the keystone species in Siberian boreal forests. Yet, despite its importance for evolutionary and population studies, the mitogenome of Siberian larch has not yet been assembled and studied.ResultsTwo sources of DNA sequences were used to search for mitochondrial DNA (mtDNA) sequences: mtDNA enriched samples and nucleotide reads generated in the de novo whole genome sequencing project, respectively. The assembly of the Siberian larch mitogenome contained nine contigs, with the shortest and the largest contigs being 24,767 bp and 4,008,762 bp, respectively. The total size of the genome was estimated at 11.7 Mbp. In total, 40 protein-coding, 34 tRNA, and 3 rRNA genes and numerous repetitive elements (REs) were annotated in this mitogenome. In total, 864 C-to-U RNA editing sites were found for 38 out of 40 protein-coding genes. The immense size of this genome, currently the largest reported, can be partly explained by variable numbers of mobile genetic elements, and introns, but unlikely by plasmid-related sequences. We found few plasmid-like insertions representing only 0.11% of the entire Siberian larch mitogenome.ConclusionsOur study showed that the size of the Siberian larch mitogenome is much larger than in other so far studied Gymnosperms, and in the same range as for the annual flowering plant Silene conica (11.3 Mbp). Similar to other species, the Siberian larch mitogenome contains relatively few genes, and despite its huge size, the repeated and low complexity regions cover only 14.46% of the mitogenome sequence.

First characterization of the complete mitochondrial genome of fungal plant-pathogen Monilinia laxa which represents the mobile intron rich structure

Monilinia laxa is an important fungal plant pathogen causing brown rot on many stone and pome fruits worldwide. Mitochondrial genome (mitogenome) plays a critical role in evolutionary biology of the organisms. This study aimed to characterize the complete mitogenome of M. laxa by using next-generation sequencing and approaches of de novo assembly and annotation. The total length of the mitogenome of M. laxa was 178,357 bp, and its structure was circular. GC content of the mitogenome was 30.1%. Annotation of the mitogenome presented 2 ribosomal RNA (rRNA) genes, 32 transfer RNA genes (tRNA), 1 gene encoding mitochondrial ribosomal protein S3, 14 protein-coding genes and 15 open reading frame encoding hypothetical proteins. Moreover, the group I mobile introns encoding homing endonucleases including LAGLIDADG and GIY-YIG families were found both within coding regions (genic) and intergenic regions of the mitogenome, indicating an enlarged size and a dynamic structure of the mitogenome. Furthermore, a comparative mitogenomic analysis was performed between M. laxa and the three closely related fungal phytopathogen species (Botryotinia fuckeliana, Sclerotinia sclerotiorum and, S. borealis). Due to the number and distribution of introns, the large extent of structural rearrangements and diverse mitogenome sizes were detected among the species investigated. Monilinia laxa presented the highest number of homing endonucleases among the fungal species considered in the analyses. This study is the first to report a detailed annotation of the mitogenome of an isolate of M. laxa, providing a solid basis for further investigations of mitogenome variations for the other Monilinia pathogens causing brown rot disease.

Intra-specific comparison of mitochondrial genomes reveals host gene fragment exchange via intron mobility in Tremella fuciformis

BackgroundMitochondrial genomic sequences are known to be variable. Comparative analyses of mitochondrial genomes can reveal the nature and extent of their variation.ResultsDraft mitochondrial genomes of 16 Tremella fuciformis isolates (TF01-TF16) were assembled from Illumina and PacBio sequencing data. Mitochondrial DNA contigs were extracted and assembled into complete circular molecules, ranging from 35,104 bp to 49,044 bp in size. All mtDNAs contained the same set of 41 conserved genes with identical gene order. Comparative analyses revealed that introns and intergenic regions were variable, whereas genic regions (including coding sequences, tRNA, and rRNA genes) were conserved. Among 24 introns detected, 11 were in protein-coding genes, 3 in tRNA genes, and the other 10 in rRNA genes. In addition, two mobile fragments were found in intergenic regions. Interestingly, six introns containing N-terminal duplication of the host genes were found in five conserved protein-coding gene sequences. Comparison of genes with and without these introns gave rise to the following proposed model: gene fragment exchange with other species can occur via gain or loss of introns with N-terminal duplication of the host genes.ConclusionsOur findings suggest a novel mechanism of fungal mitochondrial gene evolution: partial foreign gene replacement though intron mobility.

Evolution of Fusarium tricinctum and Fusarium avenaceum mitochondrial genomes is driven by mobility of introns and of a new type of palindromic microsatellite repeats

BackgroundIncreased 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.ResultsThe 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.ConclusionsMitochondrial 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.

Transitions between the steps of forward and reverse splicing of group IIC introns.

Group II introns are mobile genetic elements that perform both self-splicing and intron mobility reactions. These ribozymes are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Group IIC intron ribozymes have a minimal RNA architecture, and splice almost exclusively through hydrolysis in ribozyme reactions. Addition of the IEP allows the splicing reaction to form branched lariat RNPs capable of intron mobility. Here we examine ribozyme splicing, IEP-dependent splicing, and mobility reactions of a group IIC intron from the thermophilic bacterium Thermoanerobacter italicus (Ta.it.I1). We show that Ta.it.I1 is highly active for ribozyme activity, forming linear hydrolytic intron products. Addition of purified IEP switches activity to the canonical lariat forming splicing reaction. We demonstrate that the Ta.it.I1 group IIC intron coordinates the progression of the forward splicing reaction through a π–π′ interaction between intron domains II and VI. We further show that branched splicing is supported in the absence of the IEP when the π–π′ interaction is mutated. We also investigated the regulation of the two steps of reverse splicing during intron mobility into DNA substrates. Using a fluorescent mobility assay that simultaneously visualizes all steps of intron integration into DNA, we show that completion of reverse splicing is tightly coupled to cDNA synthesis regardless of mutation of the π–π′ interaction.

The fungal mitochondrial Nad5 pan-genic intron landscape

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.

Mitochondrial‐encoded endonucleases drive recombination of protein‐coding genes in yeast

Mitochondrial recombination in yeast is well recognized, yet the underlying genetic mechanisms are not well understood. Recent progress has suggested that mobile introns in mitochondrial genomes (mitogenomes) can facilitate the recombination of their corresponding intron-containing genes through a mechanism known as intron homing. As many mitochondrial genes lack introns, there is a critical need to determine the extent of recombination and underlying mechanism of intron-lacking genes. This study leverages yeast mitogenomes to address these questions. In Saccharomyces cerevisiae, the 3'-end sequences of at least three intron-lacking mitochondrial genes exhibit elevated nucleotide diversity and recombination hotspots. Each of these 3'-end sequences is immediately adjacent to or even fused as overlapping genes with a stand-alone endonuclease. Our findings suggest that SAEs are responsible for recombination and elevated diversity of adjacent intron-lacking genes. SAEs were also evident to drive recombination of intron-lacking genes in Lachancea kluyveri, a yeast species that diverged from S. cerevisiae more than 100 million years ago. These results suggest SAEs as a common driver in recombination of intron-lacking genes during mitogenome evolution. We postulate that the linkage between intron-lacking gene and its adjacent endonuclease gene is the result of co-evolution.