Description of Naviculavanseea sp. nov. (Naviculales, Naviculaceae), a new species of diatom from the highly alkaline Lake Van (Republic of Türkiye) with complete characterisation of its organellar genomes and multigene phylogeny.

The current article describes Naviculavanseeasp. 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 Naviculacincta and Naviculamicrodigitoradiata.

Highly Reactive Group I Introns Ubiquitous in Pathogenic Fungi.

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.

Efficient circular RNA engineering by end-to-end self-targeting and splicing reaction using Tetrahymena group I intron ribozyme.

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.

Pairwise Engineering of Tandemly Aligned Self-Splicing Group I Introns for Analysis and Control of Their Alternative Splicing.

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.

Box-shaped ribozyme octamer formed by face-to-face dimerization of a pair of square-shaped ribozyme tetramers.

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.

Comparative mitochondrial genome analyses reveal conserved gene arrangement but massive expansion/contraction in two closely related Exserohilum pathogens.

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.

RNA circles with minimized immunogenicity as potent PKR inhibitors.

Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces ∼74 nt td or ∼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.

Mitogenome-wide comparison and phylogeny reveal group I intron dynamics and intraspecific diversification within the phytopathogen Corynespora cassiicola.

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.

Effects of chain length of polyethylene glycol molecular crowders on a mutant Tetrahymena group I ribozyme lacking large peripheral module.

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+.

Organellar Introns in Fungi, Algae, and Plants.

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.

The Mitochondrial Genome of the Sea Anemone Stichodactyla haddoni Reveals Catalytic Introns, Insertion-Like Element, and Unexpected Phylogeny.

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.

Sisyphus observed: Unraveling the high ATP usage of an RNA chaperone.

DEAD-box proteins are nonprocessive RNA helicases that can function as RNA chaperones by coupling ATP binding and hydrolysis to structural reorganization of RNA. Here, Jarmoskaite et al. quantify the ATP utilization of an RNA chaperone during refolding of a misfolded ribozyme substrate. Strikingly, 100 ATP hydrolysis events are needed per successfully refolded ribozyme, suggesting that each round of unfolding requires ten ATP molecules, since 90% of substrate unfolding cycles only lead back to the kinetically favored misfolded state. This near-Sisyphean effort reveals a potentially conserved model for RNA reorganization by RNA chaperones.

No Evidence of Resistance to Trifloxystrobin, Triflumizole, and Boscalid in Podosphaera leucotricha Isolates From U.S. Commercial Apple Orchards.

Apple powdery mildew, caused by Podosphaera leucotricha, continues to be a challenge in commercial apple orchards in the U.S. Pacific Northwest and worldwide. In this study, P. leucotricha isolates were collected in 2018 and 2019 from two organic (baseline) and eight conventional (exposed) apple orchards in Washington, New York, and Virginia, and assessed for their sensitivity to trifloxystrobin (TRI, n = 232), triflumizole (TFZ, n = 217), and boscalid (BOS, n = 240) using a detached leaf assay. Effective concentrations inhibiting 50% growth (EC50) were not significantly different between baseline and exposed isolates, and ranged from 0.001 to 0.105, 0.09 to 6.31, and 0.05 to 2.18 µg/ml, for TRI, TFZ, and BOS, respectively. Reduction in sensitivity by factors of 105, 63, and 22 to TRI, TFZ, and BOS, respectively, were observed in some isolates, but all isolates were controlled by the commercial label rates of the three fungicides on detached leaves. Sequencing of the cytochrome b (cytb), cytochrome P450 sterol 14α-demethylase (CYP51), and the iron-sulfur protein subunit (SdhB) genes in isolates with high EC50 revealed no mutation previously reported to confer resistance to these fungicides in other fungi, and presence of a group I intron after codon 143 in the cytb gene. Significant (P < 0.001) moderate positive correlations (r = 0.38) observed between sensitivity to TRI and TFZ warrant continuous rotations of fungicides with different modes of action in conventional orchards. The established baseline sensitivities and the molecular markers will help in selecting discriminatory doses and bypassing the challenging in vivo testing for future sensitivity monitoring in P. leucotricha.

αßDCA method identifies unspecific binding but specific disruption of the group I intron by the StpA chaperone.

Chaperone proteins-the most disordered among all protein groups-help RNAs fold into their functional structure by destabilizing misfolded configurations or stabilizing the functional ones. But disentangling the mechanism underlying RNA chaperoning is challenging, mostly because of inherent disorder of the chaperones and the transient nature of their interactions with RNA. In particular, it is unclear how specific the interactions are and what role is played by amino acid charge and polarity patterns. Here, we address these questions in the RNA chaperone StpA. We adapted direct coupling analysis (DCA) into the αßDCA method that can treat in tandem sequences written in two alphabets, nucleotides and amino acids. With αßDCA, we could analyze StpA-RNA interactions and show consistency with a previously proposed two-pronged mechanism: StpA disrupts specific positions in the group I intron while globally and loosely binding to the entire structure. Moreover, the interactions are strongly associated with the charge pattern: Negatively charged regions in the destabilizing StpA amino-terminal affect a few specific positions in the RNA, located in stems and in the pseudoknot. In contrast, positive regions in the carboxy-terminal contain strongly coupled amino acids that promote nonspecific or weakly specific binding to the RNA. The present study opens new avenues to examine the functions of disordered proteins and to design disruptive proteins based on their charge patterns.

Catalytic RNA nano-objects formed by self-assembly of group I ribozyme dimers serving as unit structures.

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).

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

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.

Discovery of new group I-D introns leads to creation of subtypes and link to an adaptive response of the mitochondrial genome in fungi.

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.

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.

Giant group I intron in a mitochondrial genome is removed by RNA back-splicing.

BACKGROUND: The mitochondrial genomes of mushroom corals (Corallimorpharia) are remarkable for harboring two complex group I introns; ND5-717 and COI-884. How these autocatalytic RNA elements interfere with mitochondrial RNA processing is currently not known. Here, we report experimental support for unconventional processing events of ND5-717 containing RNA. RESULTS: We obtained the complete mitochondrial genome sequences and corresponding mitochondrial transcriptomes of the two distantly related corallimorpharian species Ricordea yuma and Amplexidiscus fenestrafer. All mitochondrial genes were found to be expressed at the RNA-level. Both introns were perfectly removed by autocatalytic splicing, but COI-884 excision appeared more efficient than ND5-717. ND5-717 was organized into giant group I intron elements of 18.1 kb and 19.3 kb in A. fenestrafer and R. yuma, respectively. The intron harbored almost the entire mitochondrial genome embedded within the P8 peripheral segment. CONCLUSION: ND5-717 was removed by group I intron splicing from a small primary transcript that contained a permutated intron-exon arrangement. The splicing pathway involved a circular exon-containing RNA intermediate, which is a hallmark of RNA back-splicing. ND5-717 represents the first reported natural group I intron that becomes excised by back-splicing from a permuted precursor RNA. Back-splicing may explain why Corallimorpharia mitochondrial genomes tolerate giant group I introns.

Oligomerization of a modular ribozyme assembly of which is controlled by a programmable RNA-RNA interface between two structural modules.

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.