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Branching is a critical step in RNA splicing that is essential for 5 splice site selection. Recent spliceosome structures have led to competing models for the recognition of the invariant adenosine at the branch point. However, there are no structures of any splicing complex with the adenosine nucleophile docked in the active site and positioned to attack the 5 splice site. Thus we lack a mechanistic understanding of adenosine selection and splice site recognition during RNA splicing. Here we present a cryo-electron microscopy structure of a group II intron that reveals that active site dynamics are coupled to the formation of a base triple within the branch-site helix that positions the 2-OH of the adenosine for nucleophilic attack on the 5 scissile phosphate. This structure, complemented with biochemistry and comparative analyses to splicing complexes, supports a base triple model of adenosine recognition for branching within group II introns and the evolutionarily related spliceosome.

Lycophytes and ferns represent one of the earliest-diverging lineages of vascular plants, with the Lycopodiaceae family constituting the basal clade among lycophytes. In this research, we successfully assembled and annotated the complete Lycopodium japonicum Thunb. (L. japonicum) mitochondrial genome (mitogenome) utilizing PacBio HiFi sequencing data, resulting in a single circular molecule with a size of 454,458 bp. 64 unique genes were annotated altogether, including 34 protein-coding genes, 27 tRNAs and 3 rRNAs. It also contains 32 group II introns, all of which undergo cis-splicing. We identified 195 simple sequence repeats, 1,948 dispersed repeats, and 92 tandem repeats in the L. japonicum mitogenome. Collinear analysis indicated that the mitogenomes of Lycopodiaceae are remarkably conserved compared to those of other vascular plants. We totally identified 326 RNA editing sites in 31 unique protein-coding genes with 299 sites converting cytosine to uracil and 27 sites the reverse. Notably, the L. japonicum mitogenome has small amounts foreign DNA from plastid or nuclear origin, accounting for only 2.81% of the mitogenome. The maximum likelihood phylogenetic analysis based on 23 diverse land plant mitogenomes and plastid genomes supports the basal position of lycophytes within vascular plants and they form a sister clade to all other vascular lineages, which is consistent with the PPG I classification system. As the first reported mitogenome of Lycopodioideae subfamily, this study enriches our understanding of Lycopodium mitogenomes, and sets the stage for future research on mitochondrial diversity and evolution within the lycophytes and ferns. [ABSTRACT FROM AUTHOR]

Beauveria bassiana has potential for Aedes aegypti biological control. However, its efficacy depends on the strain's geographic location, host susceptibility, and virulence. The present study aimed to evaluate the effectiveness of B. bassiana strain BBPTG4 conidia in controlling Ae. aegypti adults and its detection via introns profile on exposed mosquito corpses. Morphologic characteristics among strains were highly similar. Comprehensive testing of these strains demonstrated that BBPT4 exhibited the ideal biological activity for Ae. aegypti control, with a median lethal time (TL50) of 7.5 d compared to ~3 d and ~10 d for BB01 and BB37 strains, respectively. Infected mosquitoes died after GHA and BBPTG4 exposure, and corpses were analyzed for infecting strains detection. Differences among the seven evaluated strains were determined, assessing five different insertion group I intron profiles in BBTG4, BB01, GHA, BB37, and BB02 strains. Mosquitoes infected by BBPTG4 and non-exposed (negative control) intron profiles were obtained. We detected the presence of introns in the BBPTG4 strain, which were not present in non-exposed mosquitoes. In conclusion, B. bassiana strains showed similarities in terms of their cultural and microscopic morphological characteristics and biologicals virulence level, but different intron profiles. BBPTG4 strain-infected Ae. aegypti adult corpses, showing specific amplicons, enabled us to identify B. bassiana at the strain level among infected mosquitoes. However, monitoring and detection of field-infected insects is essential for further verification. [ABSTRACT FROM AUTHOR]

Albinism is a genetically heterogeneous disease in which 21 genes are known so far. Its inheritance mode is autosomal recessive except for one X-linked form. The molecular analysis of exonic sequences of these genes allows for about a 70% diagnostic rate. About half (15%) of the unsolved cases are heterozygous for one pathogenic or probably pathogenic variant. Assuming that the missing variant may be located in non-coding regions, we performed sequencing for 122 such heterozygous patients of either the whole genome (27 patients) or our NGS panel (95 patients) that includes, in addition to all exons of the 21 genes, the introns and flanking sequences of five genes, TYR, OCA2, SLC45A2, GPR143 and HPS1. Rare variants (MAF < 0.01) in trans to the first variant were tested by RT-PCR and/or minigene assay. Of the 14 variants tested, nine caused either exon skipping or the inclusion of a pseudoexon, allowing for the diagnosis of 11 patients. This represents 9.8% (12/122) supplementary diagnosis for formerly unsolved patients and 75% (12/16) of those in whom the candidate variant was in trans to the first variant. Of note, one missense variant was demonstrated to cause skipping of the exon in which it is located, thus shedding new light on its pathogenic mechanism. Searching for non-coding variants and testing them for an effect on RNA splicing is warranted in order to increase the diagnostic rate. [ABSTRACT FROM AUTHOR]

Background: CRISPR Cas9 and Cas12a are the two most frequently used programmable nucleases reported in plant systems. There is now a wide range of component parts for both which likely have varying degrees of effectiveness and potentially applicability to different species. Our aim was to develop and optimise Cas9 and Cas12a based systems for highly efficient genome editing in the monocotyledons barley and wheat and produce a user-friendly toolbox facilitating simplex and multiplex editing in the cereal community. Results: We identified a Zea mays codon optimised Cas9 with 13 introns in conjunction with arrayed guides driven by U6 and U3 promoters as the best performer in barley where 100% of T0 plants were simultaneously edited in all three target genes. When this system was used in wheat > 90% of T0 plants were edited in all three subgenome targets. For Cas12a, an Arabidopsis codon optimised sequence with 8 introns gave the best editing efficiency in barley when combined with a tRNA based multiguide array, resulting in 90% mutant alleles in three simultaneously targeted genes. When we applied this Cas12a system in wheat 86% & 93% of T0 plants were mutated in two genes simultaneously targeted. We show that not all introns contribute equally to enhanced mutagenesis when inserted into a Cas12a coding sequence and that there is rationale for including multiple introns. We also show that the combined effect of two features which boost Cas12a mutagenesis efficiency (D156R mutation and introns) is more than the sum of the features applied separately. Conclusion: Based on the results of our testing, we describe and provide a GoldenGate modular cloning system for Cas9 and Cas12a use in barley and wheat. Proven Cas nuclease and guide expression cassette options found in the toolkit will facilitate highly efficient simplex and multiplex mutagenesis in both species. We incorporate GRF-GIF transformation boosting cassettes in wheat options to maximise workflow efficiency. [ABSTRACT FROM AUTHOR]

Genetic features of alcohol dependence have been extensively investigated in recent years. A large body of studies has underlined the important role of genetic variants not only in metabolic pathways but also in the neurobiology of alcohol dependence, mediated by the neuronal circuits regulating reward and craving. Serotonin transporter (5-HTT), encoded by the SLC6A4 gene (Solute carrier family 6-neurotransmitter transporter-member 4), is targeted by antidepressant drugs such as selective serotonin reuptake inhibitors (SSRIs) and plays a pivotal role in serotoninergic transmission; it has been associated with psychiatric diseases and alcohol dependence. Transcriptional regulation and expression of 5-HTT depend not only on epigenetic modifications, among which DNA methylation (CpG and non-CpG) is primarily involved, but also on sequence variations occurring in intron/exon regions and in untranslated regions in 5′ and 3′, being the first sequences important for the splicing machinery and the last for the binding of transcription factors and micro RNAs. This work intends to shed light on the role of sequence variations known to affect the expression or function of 5-HTT in alcohol-dependent individuals. We found a statistically significant difference in the allelic (p = 0.0083) and genotypic (p = 0.0151) frequencies of the tri-allelic polymorphism, with higher function alleles and genotypes more represented in the control population. Furthermore, we identified three haplotypes more frequent in subjects with AUD (p < 0.0001) and one more frequent in the control population (p < 0.0001). The results obtained for the tri-allelic polymorphism in alcohol dependence confirm what is already present in part of the literature. The role of haplotypes requires further studies to be clarified. [ABSTRACT FROM AUTHOR]

This article discusses a study that explores a new strategy for constructing circular RNA (circRNA) called NeoAna, which enhances the sensitivity of non-small cell lung cancer (NSCLC) to the drug Osimertinib. The researchers successfully designed and synthesized circRNAs without scar sequences, and they observed the formation of circRNA and successful protein translation in cells. They also restored the expression of the tumor suppressor PTEN using the NeoAna system, which increased sensitivity to Osimertinib and induced apoptosis in Osimertinib-resistant cells. The researchers further demonstrated the efficacy of the NeoAna system in animal models. Overall, this study suggests that engineered circRNA can enhance the effectiveness of Osimertinib in treating NSCLC. The article also discusses the potential mechanisms of PTEN in enhancing the sensitivity of Osimertinib, a drug used to treat NSCLC. The study used RNA-seq to identify common genes between Osimertinib-resistant cells and cells with restored PTEN expression, and found that AKR1C2, a gene involved in drug resistance, was in line with the RNA-seq results. The study also explored the use of a circRNA platform called NeoAna to restore PTEN expression and overcome Osimertinib resistance, suggesting that this platform could be a promising strategy for RNA-based therapeutics. [Extracted from the article]

SUMMARY The protein levels of chloroplast photosynthetic genes and genes related to the chloroplast genetic apparatus vary to adapt to different conditions. However, the underlying mechanisms governing these variations remain unclear. The chloroplast intron Maturase K is encoded within the trnK intron and has been suggested to be required for splicing several group IIA introns, including the trnK intron. In this study, we used RNA immunoprecipitation followed by high‐throughput sequencing (RIP‐Seq) to identify MatK's preference for binding to group IIA intron domains I and VI within target transcripts. Importantly, these domains are crucial for splice site selection, and we discovered alternative 5′‐splice sites in three MatK target introns. The resulting alternative trnK lariat structure showed increased accumulation during heat acclimation. The cognate codon of tRNA‐K(UUU) is highly enriched in mRNAs encoding ribosomal proteins and a trnK‐matK over‐expressor exhibited elevated levels of the spliced tRNA‐K(UUU). Ribosome profiling analysis of the overexpressor revealed a significant up‐shift in the translation of ribosomal proteins compared to photosynthetic genes. Our findings suggest the existence of a novel regulatory mechanism linked to the abundance of tRNA‐K(UUU), enabling the differential expression of functional chloroplast gene groups. [ABSTRACT FROM AUTHOR]

The widely accepted hypothesis postulates that the first spliceosomal introns originated from group II self-splicing introns. However, it is evident that not all spliceosomal introns in the nuclear genes of modern eukaryotes are inherited through vertical transfer of intronic sequences. Several phenomena contribute to the formation of new introns but their most common origin seems to be the insertion of transposable elements. Recent analyses have highlighted instances of mass gains of new introns from transposable elements. These events often coincide with an increase or change in the spliceosome's tolerance to splicing signals, including the acceptance of noncanonical borders. Widespread acquisitions of transposon-derived introns occur across diverse evolutionary lineages, indicating convergent processes. These events, though independent, likely require a similar set of conditions. These conditions include the presence of transposon elements with features enabling their removal at the RNA level as introns and/or the existence of a splicing mechanism capable of excising unusual sequences that would otherwise not be recognized as introns by standard splicing machinery. Herein we summarize those mechanisms across different eukaryotic lineages. [ABSTRACT FROM AUTHOR]

Introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. In this work, we studied intron-encoded homing endonuclease gp210 in bacteriophage FPA3 and found that it contributes to viral competition by interfering with the replication of a coinfecting phage, φKZ. We show that gp210 targets a specific sequence in φKZ, which prevents the assembly of progeny viruses. This work demonstrates how a homing endonuclease can be deployed in interference competition among viruses and provide a relative fitness advantage. Given the ubiquity of homing endonucleases, this selective advantage likely has widespread evolutionary implications in diverse plasmid and viral competition as well as virus-host interactions. [ABSTRACT FROM AUTHOR]

Our study presents the assembly of a high-quality Taihu goose genome at the Telomere-to-Telomere (T2T) level. By employing advanced sequencing technologies, including Pacific Biosciences HiFi reads, Oxford Nanopore long reads, Illumina short reads, and chromatin conformation capture (Hi-C), we achieved an exceptional assembly. The T2T assembly encompasses a total length of 1,197,991,206 bp, with contigs N50 reaching 33,928,929 bp and scaffold N50 attaining 81,007,908 bp. It consists of 73 scaffolds, including 38 autosomes and one pair of Z/W sex chromosomes. Importantly, 33 autosomes were assembled without any gap, resulting in a contiguous representation. Furthermore, gene annotation efforts identified 34,898 genes, including 436,162 RNA transcripts, encompassing 806,158 exons, 743,910 introns, 651,148 coding sequences (CDS), and 135,622 untranslated regions (UTR). The T2T-level chromosome-scale goose genome assembly provides a vital foundation for future genetic improvement and understanding the genetic mechanisms underlying important traits in geese. [ABSTRACT FROM AUTHOR]

This article discusses a study published in Cell Proliferation that investigates the changes in mRNA isoforms during zygotic genome activation (ZGA) in mice. ZGA is the first transcriptional event after fertilization and involves the degradation of maternal mRNA and the transcription of new mRNAs from the zygotic genome. The study compares the transcriptomes of embryos with different parental genomes and stages of development to identify differential alternative splicing and alternative promoter usage events. The findings suggest that there are specific patterns of mRNA isoforms generated from the paternal and maternal genomes during ZGA, providing insights into how sperm and oocyte genomes regulate mammalian development. The study also examines the differences in mRNA isoforms before and after ZGA in mice and humans, identifying changes that can have functional implications beyond differences in mRNA abundance. The study provides insights into the regulatory mechanisms involved in early embryonic development and suggests that isoform changes can serve as markers for ZGA events, although further validation is needed to understand the specific functions of each isoform. The study was supported by various funding sources and the authors declare no conflicts of interest. [Extracted from the article]

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure. [ABSTRACT FROM AUTHOR]

Internode length is a crucial phenotypic trait of bamboo, significantly impacting its processing and utilization. Phyllostachys edulis 'Tubaeformis' (Shengyin Bamboo), a variety of Moso bamboo, exhibits drastically shortened internodes, making it a valuable ornamental bamboo species. We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin bamboo, and compared the differences between the two. Our sequencing data unveiled 139,539 AS events, with retained introns as the most prevalent events. A large number of genes were differentially alternatively spliced (DAS) between Moso bamboo and Shengyin bamboo, and genes related to RNA splicing were most significantly enriched. The high expression of SR isoforms in the 24th internode of Moso bamboo is likely the main factor leading to its greater number of alternative splicing events. Alternative splicing affects the functional domains of partial GRF, E2F, and NAM isoforms, leading to the loss of domains in some isoforms and enabling some isoforms to acquire new functional domains, and this phenomenon is more common in Shengyin bamboo. AS modifies the functional domains of certain GRF isoforms, frequently resulting in domain losses or endowing isoforms with novel domains, and this phenomenon is more common in Shengyin bamboo. We used PacBio single-molecule long-read sequencing and second-generation sequencing to identify genome-wide alternative splicing (AS) events in Moso bamboo and its dwarf mutant, Shengyin Bamboo and compared the differences between the two. [ABSTRACT FROM AUTHOR]

A single locus on the X chromosome codes for androgen receptor (AR) although this gene is subject to alternative splicing. AR is expressed in multiple tissues in males and females and is essential for reproductive success in the male. Since male and female mice are viable following naturally occurring and engineered loss of function with male mice infertile as anticipated, functional deletion of AR in pigs was hypothesized to provide a genetic containment strategy for males with edited genomes. In addition, deletion of AR might be a method to manage boar taint, hence contributing to a perceived improvement in animal welfare. The CRISPR/Cas9 technology was used to edit either exon 2 or exon 5 of the pig AR gene. Although pregnancies were established following embryo transfer of edited embryos, they were not maintained beyond day 25. Furthermore, normal M:F sex ratios were present in edited blastocysts and 19-day fetuses, but all fetuses recovered on day 21 or later were female. The pig AR gene differs from the mouse in having a U2 spliceosome component encoded in the intronic region. Hence, the absence of fetal survival beyond day 25 may be due to interference with the U2 component rather than AR.

Fluorescence in situ hybridization (FISH) is a widely used tool for quantifying gene expression and determining the location of RNA molecules in cells. We present an improved method for FISH probe production that yields high-purity probes with a wide range of fluorophores using standard laboratory equipment at low cost. The method modifies an earlier protocol that uses terminal deoxynucleotidyl transferase to add fluorescently labeled nucleotides to synthetic deoxyoligonucleotides. In our protocol, amino-11-ddUTP is joined to an oligonucleotide pool prior to its conjugation to a fluorescent dye, thereby generating pools of probes ready for a variety of modifications. This order of reaction steps allows for high labeling efficiencies regardless of the GC content or terminal base of the oligonucleotides. The degree of labeling (DOL) for spectrally distinct fluorophores (Quasar, ATTO, and Alexa dyes) was mostly >90%, comparable with commercial probes. The ease and low cost of production allowed the generation of probe sets targeting a wide variety of RNA molecules. Using these probes, FISH assays in C2C12 cells showed the expected subcellular localization of mRNAs and pre-mRNAs for Polr2a (RNA polymerase II subunit 2a) and Gapdh, and of the long noncoding RNAs Malat1 and Neat1 Developing FISH probe sets for several transcripts containing retained introns, we found that retained introns in the Gabbr1 and Noc2l transcripts are present in subnuclear foci separate from their sites of synthesis and partially coincident with nuclear speckles. This labeling protocol should have many applications in RNA biology.

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.

The self-splicing group II introns are bacterial and organellar ancestors of the nuclear spliceosome and retro-transposable elements of pharmacological and biotechnological importance. Integrating enzymatic, crystallographic, and simulation studies, we demonstrate how these introns recognize small molecules through their conserved active site. These RNA-binding small molecules selectively inhibit the two steps of splicing by adopting distinctive poses at different stages of catalysis, and by preventing crucial active site conformational changes that are essential for splicing progression. Our data exemplify the enormous power of RNA binders to mechanistically probe vital cellular pathways. Most importantly, by proving that the evolutionarily-conserved RNA core of splicing machines can recognize small molecules specifically, our work provides a solid basis for the rational design of splicing modulators not only against bacterial and organellar introns, but also against the human spliceosome, which is a validated drug target for the treatment of congenital diseases and cancers. Splicing is a vital biological reaction and a druggable pathway to treat infection, genetic diseases and cancer. Here, the authors describe how splicing is modulated by small molecules that target the conserved splicing active site in the group II introns. [ABSTRACT FROM AUTHOR]

Plant resistance against biotic stressors is significantly influenced by pathogenesis-related 1 (PR1) proteins. This study examines the systematic identification and characterization of PR1 family genes in sugarcane (Saccharum spontaneum Np-X) and the transcript expression of selected genes in two sugarcane cultivars (ROC22 and Zhongtang3) in response to Ustilago scitaminea pathogen infection. A total of 18 SsnpPR1 genes were identified at the whole-genome level and further categorized into four groups. Notably, tandem and segmental duplication occurrences were detected in one and five SsnpPR1 gene pairs, respectively. The SsnpPR1 genes exhibited diverse physio-chemical attributes and variations in introns/exons and conserved motifs. Notably, four SsnpPR1 (SsnpPR1.02/05/09/19) proteins displayed a strong protein–protein interaction network. The transcript expression of three SsnpPR1 (SsnpPR1.04/06/09) genes was upregulated by 1.2–2.6 folds in the resistant cultivar (Zhongtang3) but downregulated in the susceptible cultivar (ROC22) across different time points as compared to the control in response to pathogen infection. Additionally, SsnpPR1.11 was specifically upregulated by 1.2–3.5 folds at 24–72 h post inoculation (hpi) in ROC22, suggesting that this gene may play an important negative regulatory role in defense responses to pathogen infection. The genetic improvement of sugarcane can be facilitated by our results, which also establish the basis for additional functional characterization of SsnpPR1 genes in response to pathogenic stress. [ABSTRACT FROM AUTHOR]

Background: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns. Results: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans. Conclusions: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida. [ABSTRACT FROM AUTHOR]

Although introns are typically tens to thousands of nucleotides, there are notable exceptions. In flies as well as humans, a small number of genes contain introns that are more than 1000 times larger than typical introns, exceeding hundreds of kilobases (kb) to megabases (Mb). It remains unknown why gigantic introns exist and how cells overcome the challenges associated with their transcription and RNA processing. The Drosophila Y chromosome contains some of the largest genes identified to date: multiple genes exceed 4Mb, with introns accounting for over 99% of the gene span. Here we demonstrate that co-transcriptional splicing of these gigantic Y-linked genes is important to ensure successful transcription: perturbation of splicing led to the attenuation of transcription, leading to a failure to produce mature mRNA. Cytologically, defective splicing of the Y-linked gigantic genes resulted in disorganization of transcripts within the nucleus suggestive of entanglement of transcripts, likely resulting from unspliced long RNAs. We propose that co-transcriptional splicing maintains the length of nascent transcripts of gigantic genes under a critical threshold, preventing their entanglement and ensuring proper gene expression. Our study reveals a novel biological significance of co-transcriptional splicing. Author summary: A small number of genes in our genome are characterized by excessively large introns, sometimes exceeding megabases. It remains poorly understood why such large introns exist or how they are processed during gene expression. By examining genes on Drosophila Y chromosome that contain gigantic introns, it is shown that the transcripts of these genes are co-transcriptionally spliced, and co-transcriptional splicing of these genes is critical for proper gene expression. Perturbation of splicing led to attenuation of transcription specifically in genes with gigantic introns, but not genes with average-sized introns. Whereas transcripts of genes with gigantic introns normally form separate nuclear domains, splicing perturbation led to intermingling of these domains, leading to a proposal that co-transcriptional splicing may function to keep transcript length short to avoid entanglement of transcripts, which may stall the progression of RNA polymerase II. Taken together, these results reveal a novel function of co-transcriptional splicing that specifically impacts the expression of genes with gigantic introns. [ABSTRACT FROM AUTHOR]

Background: X-linked hypophosphatemia (XLH, OMIM 307800) is a rare phosphorus metabolism disorder caused by PHEX gene variants. Many variants simply classified as missense or nonsense variants were only analyzed at the DNA level. However, growing evidence indicates that some of these variants may alter pre-mRNA splicing, causing diseases. Therefore, this study aimed to use bioinformatics tools and a minigene assay to ascertain the effects of PHEX variations on pre-mRNA splicing. Methods: We analyzed 174 variants in the PHEX gene described as missense or nonsense variants. Finally, we selected eight candidate variants using bioinformatics tools to evaluate their effects on pre-mRNA splicing using a minigene assay system. The complementary DNA (cDNA) sequence for the PHEX gene (RefSeq NM_000444.6) serves as the basis for DNA variant numbering. Results: Of the eight candidate variants, three were found to cause abnormal splicing. Variants c.617T>G p.(Leu206Trp) and c.621T>A p.(Tyr207*) in exon 5 altered the splicing of pre-mRNA, owing to the activation of a cryptic splice site in exon 5, which produced an aberrant transcript lacking a part of exon 5, whereas variant c.1700G>C p.(Arg567Pro) in exon 16 led to the activation of a cryptic splice site in intron 16, resulting in a partial inclusion of intron 16. Conclusion: Our study employed a minigene system, which has a great degree of flexibility to assess abnormal splicing patterns under the circumstances of patient mRNA samples that are not available, to explore the impact of the exonic variants on pre-mRNA splicing. Based on the aforementioned experimental findings, we demonstrated the importance of analyzing exonic variants at the mRNA level. [ABSTRACT FROM AUTHOR]

Background: The disputed phylogenetic position of Aerides flabellata Rolfe ex Downie, due to morphological overlaps with related species, was investigated based on evidence of complete chloroplast (cp) genomes. The structural characterization of complete cp genomes of A. flabellata and A. rosea Lodd. ex Lindl. & Paxton were analyzed and compared with those of six related species in "Vanda-Aerides alliance" to provide genomic information on taxonomy and phylogeny. Results: The cp genomes of A. flabellata and A. rosea exhibited conserved quadripartite structures, 148,145 bp and 147,925 bp in length, with similar GC content (36.7 ~ 36.8%). Gene annotations revealed 110 single-copy genes, 18 duplicated in inverted regions, and ten with introns. Comparative analysis across related species confirmed stable sequence identity and higher variation in single-copy regions. However, there are notable differences in the IR regions between two Aerides Lour. species and the other six related species. The phylogenetic analysis based on CDS from complete cp genomes indicated that Aerides species except A. flabellata formed a monophyletic clade nested in the subtribe Aeridinae, being a sister group to Renanthera Lour., consistent with previous studies. Meanwhile, a separate clade consisted of A. flabellata and six Vanda R. Br. species was formed, as a sister taxon to Holcoglossum Schltr. Conclusions: This research was the first report on the complete cp genomes of A. flabellata. The results provided insights into understanding of plastome evolution and phylogenetic relationships of Aerides. The phylogenetic analysis based on complete cp genomes showed that A. flabellata should be placed in Vanda rather than in Aerides. [ABSTRACT FROM AUTHOR]

Summary: Most splicing factors are extensively phosphorylated but their physiological functions in plant salt resistance are still elusive.We found that phosphorylation by SnRK1 kinase is essential for SRRM1L nuclear speckle formation and its splicing factor activity in plant cells. In Arabidopsis, loss‐of‐function of SRRM1L leads to the occurrence of alternative pre‐mRNA splicing events and compromises plant resistance to salt stress.In Arabidopsis srrm1l mutant line, we identified an intron‐retention Nuclear factor Y subunit A 10 (NFYA10) mRNA variant by RNA‐Seq and found phosphorylation‐dependent RNA‐binding of SRRM1L is indispensable for its alternative splicing activity. In the wild‐type Arabidopsis, salt stress can activate SnRK1 to phosphorylate SRRM1L, triggering enrichment of functional NFYA10.1 variant to enhance plant salt resistance. By contrast, the Arabidopsis srrm1l mutant accumulates nonfunctional NFYA10.3 variant, sensitizing plants to salt stress.In summary, this work deciphered the molecular mechanisms and physiological functions of SnRK1‐SRRM1L‐NFYA10 module, shedding light on a regulatory pathway to fine‐tune plant adaptation to abiotic stress at the post‐transcriptional and post‐translational levels. [ABSTRACT FROM AUTHOR]

Genome size varies greatly across the tree of life and transposable elements are an important contributor to this variation. Among vertebrates, amphibians display the greatest variation in genome size, making them ideal models to explore the causes and consequences of genome size variation. However, high-quality genome assemblies for amphibians have, until recently, been rare. Here, we generate a high-quality genome assembly for the dyeing poison frog, Dendrobates tinctorius. We compare this assembly to publicly available frog genomes and find evidence for both large-scale conserved synteny and widespread rearrangements between frog lineages. Comparing conserved orthologs annotated in these genomes revealed a strong correlation between genome size and gene size. To explore the cause of gene-size variation, we quantified the location of transposable elements relative to gene features and find that the accumulation of transposable elements in introns has played an important role in the evolution of gene size in D. tinctorius , while estimates of insertion times suggest that many insertion events are recent and species-specific. Finally, we carry out population-scale mobile-element sequencing and show that the diversity and abundance of transposable elements in poison frog genomes can complicate genotyping from repetitive element sequence anchors. Our results show that transposable elements have clearly played an important role in the evolution of large genome size in D. tinctorius. Future studies are needed to fully understand the dynamics of transposable element evolution and to optimize primer or bait design for cost-effective population-level genotyping in species with large, repetitive genomes. [ABSTRACT FROM AUTHOR]

Gene disruption from double‐strand DNA breaks within introns is a mechanism of inactivating the tumor suppressor TP53. This occurs more frequently in osteosarcoma and biliary adenocarcinoma compared with other cancer types. The patterns of intron breakpoints within TP53 do not correlate with prevalence, intron length, or overall genome‐wide levels of rearrangements. Therefore, these breakpoints appear to be selected for reasons other than to disrupt TP53. A recent article published by Saba et al in The Journal of Pathology illustrates a benefit to having breakpoints within intron 1 using high‐quality matched genomic and transcriptomic osteosarcoma sequencing data as well as in vitro validation. The authors describe how the rearrangement results in relocation of the TP53 promoter region to regions upstream of genes that encode members of cartilage, growth plate development, osteoclast formation, and other TP53‐related pathways. The upregulation of these genes by the TP53 promoter are gain‐of‐function events that are likely to promote tumor development and growth. Therefore, this article presents a potential new paradigm in which a single mutation would result in both the loss of a tumor suppressor and the gain of an oncogenic program. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Background: Eukaryotic genes contain introns that are removed by the spliceosomal machinery during mRNA maturation. Introns impose a huge energetic burden on a cell; therefore, they must play an essential role in maintaining genome stability and/or regulating gene expression. Many genes (> 50%) in Plasmodium parasites contain predicted introns, including introns in 5′ and 3′ untranslated regions (UTR). However, the roles of UTR introns in the gene expression of malaria parasites remain unknown. Methods: In this study, an episomal dual-luciferase assay was developed to evaluate gene expression driven by promoters with or without a 5′UTR intron from four Plasmodium yoelii genes. To investigate the effect of the 5′UTR intron on endogenous gene expression, the pytctp gene was tagged with 3xHA at the N-terminal of the coding region, and parasites with or without the 5′UTR intron were generated using the CRISPR/Cas9 system. Results: We showed that promoters with 5′UTR introns had higher activities in driving gene expression than those without 5′UTR introns. The results were confirmed in recombinant parasites expressing an HA-tagged gene (pytctp) driven by promoter with or without 5′UTR intron. The enhancement of gene expression was intron size dependent, but not the DNA sequence, e.g. the longer the intron, the higher levels of expression. Similar results were observed when a promoter from one strain of P. yoelii was introduced into different parasite strains. Finally, the 5′UTR introns were alternatively spliced in different parasite development stages, suggesting an active mechanism employed by the parasites to regulate gene expression in various developmental stages. Conclusions: Plasmodium 5′UTR introns enhance gene expression in a size-dependent manner; the presence of alternatively spliced mRNAs in different parasite developmental stages suggests that alternative slicing of 5′UTR introns is one of the key mechanisms in regulating parasite gene expression and differentiation. [ABSTRACT FROM AUTHOR]

Exons crucial for coding are often hidden within introns, and the two tend to vary greatly in length, which results in deep learning-based protein coding region prediction methods often performing poorly when applied to more structurally complex biological genomes. DNA shape information also plays a role in revealing the underlying logic of gene expression, yet current methods ignore the influence of DNA shape features when distinguishing coding and non-coding regions. We propose a method to predict protein-coding regions using the CNNS-BRNN model, which incorporates DNA shape features and improves the model's ability to distinguish between intronic and exonic features. We use a fusion coding technique that combines DNA shape features and traditional sequence features. Experiments show that this method outperforms the baseline method in metrics such as AUC and F1 by 2.3% and 5.3%, respectively, and the fusion coding method that introduces DNA shape features has a significant improvement in model performance. [ABSTRACT FROM AUTHOR]

Pre-mRNA splicing is a fundamental step in gene expression, conserved across eukaryotes, in which the spliceosome recognizes motifs at the 3' and 5' splice sites (SSs), excises introns, and ligates exons. SS recognition and pairing is often influenced by protein splicing factors (SFs) that bind to splicing regulatory elements (SREs). Here, we describe SMsplice, a fully interpretable model of pre-mRNA splicing that combines models of core SS motifs, SREs, and exonic and intronic length preferences. We learn models that predict SS locations with 83 to 86% accuracy in fish, insects, and plants and about 70% in mammals. Learned SRE motifs include both known SF binding motifs and unfamiliar motifs, and both motif classes are supported by genetic analyses. Our comparisons across species highlight similarities between non-mammals, increased reliance on intronic SREs in plant splicing, and a greater reliance on SREs in mammalian splicing. [ABSTRACT FROM AUTHOR]

A new Cephalaria (Caprifoliaceae) species, Cephalaria gokturkii, is described and illustrated from Girdev Mountain (Muğla Province) for the Flora of Türkiye. Throughout this manuscript, the new species and its relatives were compared in terms of morphological, palynological and molecular features. The obtained findings indicated that the new species is similar to C. elmaliensis but is characterized by having larger capitula and receptacular bracts in flower (vs. 1-2 cm capitula and less than 3 mm in flower) and hairy trigonous-narrowly ovate involucral bracts (vs. glabrous and broadly ovate involucral bracts) and smaller involucels (vs. involucels longer than 10 mm in fruit). Although the new species is similar to C. elmaliensis in terms of pollen shape and general appearance, its pollens are larger in terms of mean equatorial diameter and polar axis. Following morphological data, molecular analyses (based on ITS and trnL introns) indicated that the phylogenetically closest relative of the species is also C. elmaliensis. Additionally, we report that taxonomic notes for this new species are provided, including distribution, ecology, and conservation status. [ABSTRACT FROM AUTHOR]

The availability of an alternative and efficient genetic editing technology is critical for fundamental research and strain improvement engineering of Streptomyces species, which are prolific producers of complex secondary metabolites with significant pharmaceutical activities. The mobile group II introns are retrotransposons that employ activities of catalytic intron RNAs and intron‐encoded reverse transcriptase to precisely insert into DNA target sites through a mechanism known as retrohoming. We here developed a group II intron‐based gene editing tool to achieve precise chromosomal gene insertion in Streptomyces. Moreover, by repressing the potential competition of RecA‐dependent homologous recombination, we enhanced site‐specific insertion efficiency of this tool to 2.38%. Subsequently, we demonstrated the application of this tool by screening and characterizing the secondary metabolite biosynthetic gene cluster (BGC) responsible for synthesizing the red pigment in Streptomyces roseosporus. Accompanied with identifying and inactivating this BGC, we observed that the impair of this cluster promoted cell growth and daptomycin production. Additionally, we applied this tool to activate silent jadomycin BGC in Streptomyces venezuelae. Overall, this work demonstrates the potential of this method as an alternative tool for genetic engineering and cryptic natural product mining in Streptomyces species. [ABSTRACT FROM AUTHOR]

Turhadi, Sudarjayanti BN, Solihah FM, Azrianingsih R, Afiyanti M, Arumingtyas EL. 2024. Molecular identification of Scopellaria marginata from East Java, Indonesia, based on trnL-UAA and trnL-trnF intergenic spacer regions. Nusantara Bioscience 16: 111-118. Scopellaria marginata (Blume) W.J.de Wilde & Duyfjes is a wild species in Cucurbitaceae, which was recorded as new expanding their distribution in East Java, Indonesia. The trnL-UAA and trnL-trnF intergenic spacer (IGS) sequences of Scopellaria are still limited in publicly accessible databases. This study aimed to evaluate the trnL-UAA and trnL-trnF IGS sequences of S. marginata from Malang, East Java. Total DNA of S. marginata was used to amplify the trnL-UAA and trnL-trnF IGS region. Then, the PCR products are sequenced using bi-directional Sanger dideoxy sequencing to obtain the DNA sequence of those two regions. The results showed that the partial sequences of S. marginata for trnL-UAA ranged from 528 to 571 bp, while the sequence for trnF-trnL IGS ranged from 434 to 445 bp. The S. marginata samples are similar to S. marginata in the database with similarity levels of 97.14-97.50% and 98.36-98.61%, respectively, based on the trnL-UAA and trnL-trnF IGS. Both trnL-UAA and trnL-trnF IGS showed a Correct Assignment Rate (CAR) of 100% for S. marginata. Two-dimensional DNA barcoding with lengths 505 and 417 bp for trnL-UAA and trnL-trnF IGS proposed as specific barcodes for S. marginata. These results prove that Malang, East Java was an additional distribution area for S. marginata in Indonesia. [ABSTRACT FROM AUTHOR]

Fryns syndrome (FS) is a multiple congenital anomaly syndrome with different multisystemic malformations. These include congenital diaphragmatic hernia, pulmonary hypoplasia, and craniofacial dysmorphic features in combination with malformations of the central nervous system such as agenesis of the corpus callosum, cerebellar hypoplasia, and enlarged ventricles. We present a non-consanguineous northern European family with two recurrent cases of FS: a boy with multiple congenital malformations who died at the age of 2.5 months and a female fetus with a complex developmental disorder with similar features in a following pregnancy. Quad whole exome analysis revealed two likely splicing-affecting disease-causing mutations in the PIGN gene: a synonymous mutation c.2619G>A, p.(Leu873=) in the last nucleotide of exon 29 and a 30 bp-deletion c.996_1023+2del (NM_176787.5) protruding into intron 12, with both mutations in trans configuration in the affected patients. Exon skipping resulting from these two variants was confirmed via RNA sequencing. Our molecular and clinical findings identified compound heterozygosity for two novel splice-affecting variants as the underlying pathomechanism for the development of FS in two patients. [ABSTRACT FROM AUTHOR]

Objective To identify the TMPRSS6 gene variants in Mexican patients with iron treatment refractoriness, to describe hematological and iron profile parameters, and to use bioinformatic prediction and protein modeling tools to assess a possible biological impact for the detected missense variants. Methods Nineteen patients referred with iron treatment refractoriness were studied. Peripheral blood was collected to determine hematic cytometry, iron profile, hemoglobin electrophoresis, and quantification. Molecular screening was carried out for exons 15 through 18 of the TMPRSS6 gene by Sanger sequencing and for frequent thalassemia variants by amplification-refractory mutation system-polymerase chain reaction (PCR) and gap-PCR. The biological impact of the detected missense variants was assessed using bioinformatic prediction and protein modeling tools. Results We found 5 genetic variants in the matriptase-2 catalytic domain: 1 at intron-15/exon-16 junction (rs60484081) and 4 exonic, 3 missense (rs377054987, p.Gly626Asp; rs1384127820, p.Ser672Thr; rs855791, p.Val727Ala) and 1 synonymous (rs2235321, p.Tyr730=), with frequencies ranging from 0.18 to 0.53. No significant differences were observed in the hematological parameters or iron profile, considering type and number of variants. Bioinformatic predictions suggested a possible biological impact only for rs377054987. Conclusions The TMPRSS6 variants observed in Mexican patients with oral iron treatment refractoriness have high frequencies; nevertheless, their relationship with hematological and iron profile parameters needs further research. The possible biological impact for rs377054987 is due to size and amino acid hydrophobicity changes and hydrogen bond modifications. [ABSTRACT FROM AUTHOR]

Congenital contractural arachnodactyly (CCA) is an extremely rare autosomal dominant connective tissue genetic disorder caused by pathogenic variants in FBN2. CCA is characterized by arachnodactyly, camptodactyly, contracture of major joints, scoliosis, pectus deformities, and crumpled ears, but rarely with lethal cardiovascular manifestations as in Marfan syndrome. It is imperative to conduct a comprehensive analysis and review of the pathogenesis of CCA resulting from pathogenic variants in FBN2 gene. Using whole-exome sequencing and Sanger sequencing, we identified a novel pathogenic splice-altering variant (c.4472-3C>A) in intron 34 of FBN2 gene in a CCA pedigree. The transcriptional result of the splicing-altering variant was analyzed by RNA sequencing. We systematically analyzed the clinical manifestations of all reported cases of CCA caused by splicing-altering pathogenic variants and focused on all the pathogenic variants in FBN2 gene that are associated with severe cardiovascular manifestations. The splice-altering variant (c.4472-3C>A) in FBN2 was demonstrated to result in the exon 35 skipping and cause an in-frame deletion. Furthermore, we identified exons 31 to 35 may be a hotspot region in FBN2 gene associated with severe cardiovascular phenotype. This study enriched the pathogenic spectrum of CCA and identified a hotspot region in FBN2 gene associated with severe cardiovascular manifestations. We recommend that patients carrying pathogenic variants in exons 31 to 35 of FBN2 pay more attention to cardiac evaluation. [ABSTRACT FROM AUTHOR]

It has been the aim of this study to molecular-taxonomically identify 15 Beauveria isolates collected from different geographical regions and insect hosts in Argentina and to investigate the levels of inter- and intra-specific diversity across this set of isolates. Based on phylogenetic analyses of EF1A-RPB1-RPB2 concatenated genes and BLOC markers, all Beauveria strains were identify as Beauveria bassiana. Within the B. bassiana clades of both phylogenies, isolates from Argentina were not clustered according to geographic origin or host. The 15 fungal isolates were further analyzed by PCR amplification of the intron insertion hot spot region of the nuclear 28S rRNA encoding sequence. By intron sequence and position, seven different group-I intron combinations termed variants A, B1, B2, C, D, E and F were found in the 15 isolates under study. Variants B1/B2 consisting of a single 28Si2 intron were found in ten isolates, whereas variant A occurred twice and variants C through F were unique across the set of isolates under study. The determination of the different introns and intron combinations in the 28S rRNA gene is a powerful tool for achieving infraspecific differentiation of B. bassiana isolates from Argentina. • All Beauveria strains from Argentina were identify as B. bassiana. • Seven group-I intron combinations were found in the 15 B. bassiana isolates. • Introns in 28S rRNA gene is useful for infraspecific differentiation of B. bassiana. [ABSTRACT FROM AUTHOR]

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Background: Genomic architecture is a key evolutionary trait for living organisms. Due to multiple complex adaptive and neutral forces which impose evolutionary pressures on genomes, there is a huge variability of genomic features. However, their variability and the extent to which genomic content determines the distribution of recovered loci in reduced representation sequencing studies is largely unexplored. Results: Here, by using 80 genome assemblies, we observed that whereas plants primarily increase their genome size by expanding their intergenic regions, animals expand both intergenic and intronic regions, although the expansion patterns differ between deuterostomes and protostomes. Loci mapping in introns, exons, and intergenic categories obtained by in silico digestion using 2b-enzymes are positively correlated with the percentage of these regions in the corresponding genomes, suggesting that loci distribution mostly mirrors genomic architecture of the selected taxon. However, exonic regions showed a significant enrichment of loci in all groups regardless of the used enzyme. Moreover, when using selective adaptors to obtain a secondarily reduced loci dataset, the percentage and distribution of retained loci also varied. Adaptors with G/C terminals recovered a lower percentage of selected loci, with a further enrichment of exonic regions, while adaptors with A/T terminals retained a higher percentage of loci and slightly selected more intronic regions than expected. Conclusions: Our results highlight how genome composition, genome GC content, RAD enzyme choice and use of base-selective adaptors influence reduced genome representation techniques. This is important to acknowledge in population and conservation genomic studies, as it determines the abundance and distribution of loci. [ABSTRACT FROM AUTHOR]

Understanding the forces that shape population genetic structure is fundamental both for understanding evolutionary trajectories and for conservation. Many factors can influence the geographic distribution of genetic variation, and the extent to which local populations differ can be especially difficult to predict in highly mobile organisms. For example, many species of seabirds are essentially panmictic, but some show strong structure. Pigeon Guillemots (Cepphus columba; Charadriiformes: Alcidae) breed in small colonies scattered along the North Pacific coastline and feed in shallow nearshore waters year‐round. Given their distribution, gene flow is potentially lower and population genetic structure is stronger than in most other high‐latitude Northern Hemisphere seabirds. We screened variation in the mitochondrial control region, four microsatellite loci, and two nuclear introns in 202 Pigeon Guillemots representing three of five subspecies. Mitochondrial sequences and nuclear loci both showed significant population differences, although structure was weaker for the nuclear loci. Genetic differentiation was correlated with geographic distance between sampling locations for both the mitochondrial and nuclear loci. Mitochondrial gene trees and demographic modeling both provided strong evidence for two refugial populations during the Pleistocene glaciations: one in the Aleutian Islands and one farther east and south. We conclude that historical fragmentation combined with a stepping‐stone model of gene flow led to the relatively strong population differentiation in Pigeon Guillemots compared to other high‐latitude Northern Hemisphere seabird species. Our study adds to growing evidence that Pleistocene glaciation events affected population genetic structure not only in terrestrial species but also in coastal marine animals. [ABSTRACT FROM AUTHOR]

This article discusses the potential of Stathmin 2 (STMN2) as a treatment target for TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS). TDP-43 is a protein associated with ALS, and its abnormal modifications lead to neurotoxic effects. The study found that the expression of STMN2 was significantly reduced in the presence of TDP-43 depletion, and restoring STMN2 levels rescued neurite outgrowth and axon regeneration. The article suggests that STMN2 is involved in ALS pathology and could be a promising therapeutic approach. However, further research and in vivo examination are needed to confirm the efficacy and safety of targeting STMN2. [Extracted from the article]

Circular RNAs (circRNAs), are a covalently closed, single-stranded RNA without 5′- and 3′-termini, commonly stem from the exons of precursor mRNAs (pre-mRNAs). They have recently garnered interest, with studies uncovering their pivotal roles in regulating various aspects of cell functions and disease progressions. A notable feature of circRNA lies in the mechanism of its biogenesis involving a specialized form of splicing: back-splicing. A splicing process that relies on interactions between introns flanking the circularizing exon to bring the up and downstream splice sites in proximity through the formation of a prerequisite hairpin structure, allowing the spliceosomes to join the two splice sites together to produce a circular RNA molecule. Based on this mechanism, we explored the feasibility of facilitating the formation of such a prerequisite hairpin structure by utilizing a newly designed oligonucleotide, CircuLarIzation Promoting OligoNucleotide (CLIP-ON), to promote the production of circRNA in cells. CLIP-ON was designed to hybridize with and physically bridge two distal sequences in the flanking introns of the circularizing exons. The feasibility of CLIP-ON was confirmed in HeLa cells using a model pre-mRNA, demonstrating the applicability of CLIP-ON as a trans-acting modulator to upregulate the production of circRNAs in a cellular environment. [ABSTRACT FROM AUTHOR]

Splicing is the stepwise molecular process by which introns are removed from premRNA and exons are joined together to form mature mRNA sequences. The ordering and spatial distribution of these steps remain controversial, with opposing models suggesting splicing occurs either during or after transcription. We used single-molecule RNA FISH, expansion microscopy, and live-cell imaging to reveal the spatiotemporal distribution of nascent transcripts in mammalian cells. At super-resolution levels, we found that pre-mRNA formed clouds around the transcription site. These clouds indicate the existence of a transcription-site-proximal zone through which RNA move more slowly than in the nucleoplasm. Full-length pre-mRNA undergo continuous splicing as they move through this zone following transcription, suggesting a model in which splicing can occur post-transcriptionally but still within the proximity of the transcription site, thus seeming co-transcriptional by most assays. These results may unify conflicting reports of co-transcriptional versus post-transcriptional splicing. [ABSTRACT FROM AUTHOR]

Aging and senescence are characterized by pervasive transcriptional dysfunction, including increased expression of transposons and introns. Our aim was to elucidate mechanisms behind this increased expression. Most transposons are found within genes and introns, with a large minority being close to genes. This raises the possibility that transcriptional readthrough and intron retention are responsible for age- related changes in transposon expression rather than expression of autonomous transposons. To test this, we compiled public RNA- seq datasets from aged human fibroblasts, replicative and drug- induced senescence in human cells, and RNA- seq from aging mice and senescent mouse cells. Indeed, our reanalysis revealed a correlation between transposons expression, intron retention, and transcriptional readthrough across samples and within samples. Both intron retention and readthrough increased with aging or cellular senescence and these transcriptional defects were more pronounced in human samples as compared to those of mice. In support of a causal connection between readthrough and transposon expression, analysis of models showing induced transcriptional readthrough confirmed that they also show elevated transposon expression. Taken together, our data suggest that elevated transposon reads during aging seen in various RNA- seq dataset are concomitant with multiple transcriptional defects. Intron retention and transcriptional readthrough are the most likely explanation for the expression of transposable elements that lack a functional promoter. [ABSTRACT FROM AUTHOR]

SOSs are key genes in the SOS (salt overly sensitive) signaling pathway, which plays an important role in maintaining ion homeostasis in plants under salt stress. Our aim was to clarify the biological function of the SOS gene family in tomato plants. We identified 14 SpeSOS genes, 10 SpiSOS genes, 11 SpmSOS genes, 9 SlmSOS genes, and 11 SlySOS genes from the genomes of "LA0716" (Spe), "LA2093" (Spi), "LA1589" (Spm), "M82" (Slm), and "Heinz 1706" (Sly) separately. The SOS protein family in tomatoes was divided into five subgroups (SOS1, SOS2, SOS3, SOS4, and SOS5) through phylogenetic analysis. The SOS proteins of the same subgroup in tomatoes contained similar conserved domains and motif structures. A subcellular localization prediction showed that the SOS1, SOS3, and SOS5 proteins in tomatoes were located on the cell membrane, while the SOS2 and SOS4 proteins in tomatoes were located on the cytoplasm and chloroplast, respectively. SlSOS1 contained the most exons and introns (23 and 22, respectively), while SlSOS5 contained only one exon. Via the analysis of the cis-elements in the promoters of those SOS genes in tomatoes, several hormone-, light-, and abiotic stress-related cis-elements were found. In addition, qRT-PCR revealed that the SpeSOS, SpiSOS, and SlySOS genes were induced by salt stress with similar expression patterns. Additionally, the expressions of SOS1-1, SOS1-2, SOS2-2, SOS3-3, SOS4-1, and SOS5-2 were higher in salt-tolerant tomatoes compared with salt-sensitive tomatoes under salt stress. In the salt-sensitive "LA1698" tomato and salt-tolerant "LA0516" tomato, most SOS genes had the highest expression in the roots. The expressions of SOS1-1, SOS1-2, SOS2-1, SOS2-2, SOS3-2, SOS3-3, and SOS5-1 in the leaves of salt-tolerant tomatoes were significantly higher than those in salt-sensitive tomatoes. Thereby, the SOS genes in tomatoes were induced by salt stress, indicating that they participated in the regulation mechanism of tomato salt tolerance. This study laid the foundation for further study on the function of the SOS gene family and revealed the molecular mechanism of tomato salt resistance. [ABSTRACT FROM AUTHOR]

Two well-known facets in protein synthesis in eukaryotic cells are transcription of DNA to pre-RNA in the nucleus and the translation of messenger-RNA (mRNA) to proteins in the cytoplasm. A critical intermediate step is the removal of segments (introns) containing ∼ 97 % of the nucleic-acid sites in pre-RNA and sequential alignment of the retained segments (exons) to form mRNA through a process referred to as splicing. Alternative forms of splicing enrich the proteome while abnormal splicing can enhance the likelihood of a cell developing cancer or other diseases. Mechanisms for splicing and origins of splicing errors are only partially deciphered. Our goal is to determine if rules on splicing can be inferred from data analytics on nucleic-acid sequences. Toward that end, we represent a nucleic-acid site as a point in a plane defined in terms of the anterior and posterior sub-sequences of the site. The "point-set" representation expands analytical approaches, including the use of statistical tools, to characterize genome sequences. It is found that point-sets for exons and introns are visually different, and that the differences can be quantified using a family of generalized moments. We design a machine-learning algorithm that can recognize individual exons or introns with 91 % accuracy. Point-set distributions and generalized moments are found to differ between organisms. [ABSTRACT FROM AUTHOR]

Population genetic inference of selection on the nucleotide sequence level often proceeds by comparison to a reference sequence evolving only under mutation and population demography. Among the few candidates for such a reference sequence is the 5' part of short introns (5SI) in Drosophila. In addition to mutation and population demography, however, there is evidence for a weak force favouring GC bases, likely due to GC -biased gene conversion (gBGC), and for the effect of linked selection. Here, we use polymorphism and divergence data of Drosophila melanogaster to detect and describe the forces affecting the evolution of the 5SI. We separately analyse mutation classes, compare them between chromosomes, and relate them to recombination rate frequencies. GC -conservative mutations seem to be mainly influenced by mutation and drift, with linked selection mostly causing differences between the central and the peripheral (i.e. telomeric and centromeric) regions of the chromosome arms. Comparing GC -conservative mutation patterns between autosomes and the X chromosome showed differences in mutation rates, rather than linked selection, in the central chromosomal regions after accounting for differences in effective population sizes. On the other hand, GC -changing mutations show asymmetric site frequency spectra, indicating the presence of gBGC, varying among mutation classes and in intensity along chromosomes, but approximately equal in strength in autosomes and the X chromosome. [ABSTRACT FROM AUTHOR]

Cellular respiration involves complex organellar metabolic activities that are pivotal for plant growth and development. Mitochondria contain their own genetic system (mitogenome, mtDNA), which encodes key elements of the respiratory machinery. Plant mtDNAs are notably larger than their counterparts in Animalia, with complex genome organization and gene expression characteristics. The maturation of the plant mitochondrial transcripts involves extensive RNA editing, trimming and splicing events. These essential processing steps rely on the activities of numerous nuclear-encoded cofactors, which may also play key regulatory roles in mitochondrial biogenesis and function and hence in plant physiology. Proteins that harbor the plant organelle RNA recognition (PORR) domain are represented in a small gene family in plants. Several PORR members, including WTF1, WTF9 and LEFKOTHEA, are known to act in the splicing of organellar group II introns in angiosperms. The AT4G33495 gene locus encodes an essential PORR protein in Arabidopsis , termed ROOT PRIMORDIUM DEFECTIVE 1 (RPD1). A null mutation of At.RPD1 causes arrest in early embryogenesis, while the missense mutant lines, rpd1.1 and rpd1.2 , exhibit a strong impairment in root development and retarded growth phenotypes, especially under high-temperature conditions. Here, we further show that RPD1 functions in the splicing of introns that reside in the coding regions of various complex I (CI) subunits (i.e. nad2, nad4, nad5 and nad7), as well as in the maturation of the ribosomal rps3 pre-RNA in Arabidopsis mitochondria. The altered growth and developmental phenotypes and modified respiration activities are tightly correlated with respiratory chain CI defects in rpd1 mutants. [ABSTRACT FROM AUTHOR]

The article describes the case of a 4-year-old girl which illustrates the biallelic variants in RINT1 should be considered in children presenting with pure and complex hereditary spastic paraplegias. Topics covered include the symptoms and clinical features, the experiments in patient fibroblasts conducted and the need for future research in prospective longitudinal natural history studies.

Alternative splicing enables higher eukaryotes to expand mRNA diversity from a finite number of genes through highly combinatorial splice site selection mechanisms that are influenced by the sequence of competing splice sites, cis-regulatory elements binding trans-acting factors, the length of exons and introns harbouring alternative splice sites and RNA secondary structures at putative splice junctions. To test the hypothesis that the intron definition or exon definition modes of splice site recognition direct the selection of alternative splice patterns, we created a database of alternative splice site usage (ALTssDB). When alternative splice sites are embedded within short introns (intron definition), the 5' and 3' splice sites closest to each other across the intron preferentially pair, consistent with previous observations. However, when alternative splice sites are embedded within large flanking introns (exon definition), the 5' and 3' splice sites closest to each other across the exon are preferentially selected. Thus, alternative splicing decisions are influenced by the intron and exon definition modes of splice site recognition. The results demonstrate that the spliceosome pairs splice sites that are closest in proximity within the unit of initial splice site selection.

There is massive variation in intron numbers across eukaryotic genomes, yet the major drivers of intron content during evolution remain elusive. Rapid intron loss and gain in some lineages contrast with long-term evolutionary stasis in others. Episodic intron gain could be explained by recently discovered specialized transposons called Introners, but so far Introners are only known from a handful of species. Here, we performed a systematic search across 3,325 eukaryotic genomes and identified 27,563 Introner-derived introns in 175 genomes (5.2%). Species with Introners span remarkable phylogenetic diversity, from animals to basal protists, representing lineages whose last common ancestor dates to over 1.7 billion years ago. Aquatic organisms were 6.5 times more likely to contain Introners than terrestrial organisms. Introners exhibit mechanistic diversity but most are consistent with DNA transposition, indicating that Introners have evolved convergently hundreds of times from nonautonomous transposable elements. Transposable elements and aquatic taxa are associated with high rates of horizontal gene transfer, suggesting that this combination of factors may explain the punctuated and biased diversity of species containing Introners. More generally, our data suggest that Introners may explain the episodic nature of intron gain across the eukaryotic tree of life. These results illuminate the major source of ongoing intron creation in eukaryotic genomes.