Intron Sequences Research Articles

Multiple losses of aKRAB from PRDM9 coincide with a teleost-specific intron size distribution

BackgroundPrimary transcripts are largely comprised of intronic sequences that are excised and discarded shortly after synthesis. In vertebrates, the shape of the intron size distribution is largely constant; however, most teleost fish have a diverged log-bimodal ‘teleost distribution’ (TD) that is seen only in teleosts. How the TD evolved and to what extent this was affected by adaptative or non-adaptive mechanisms is unknown.ResultsHere, we show that the TD has evolved independently at least six times and that its appearance is linked to the loss of the aKRAB domain from PRDM9. We determined intron size distributions and identified PRDM9 orthologues from annotated genomes in addition to scanning 1193 teleost assemblies for the aKRAB domain. We show that a diverged form of PRDM9 (β\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\beta$$\\end{document}) is predominant in teleosts whereas the α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document} version is absent from most species. Only a subset of PRDM9-α\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\alpha$$\\end{document} proteins contain aKRAB, and hence, it is present only in a small number of teleost lineages. Almost all lineages lacking aKRAB (but no species with) had TDs.ConclusionsIn mammals, PRDM9 defines the sites of meiotic recombination through a mechanism that increases structural variance and depends on aKRAB. The loss of aKRAB is likely to have shifted the locations of both recombination and structural variance hotspots. Our observations suggest that the TD evolved as a side-effect of these changes and link recombination to the evolution of intron size illustrating how genome architectures can evolve in the absence of selection.

OncoSplicing 3.0: an updated database for identifying RBPs regulating alternative splicing events in cancers.

Alternative splicing (AS) is a crucial mechanism to regulate gene expression and protein complexity. RNA-binding proteins (RBPs) play an important role in regulating abnormal alternative splicing in cancers. However, few resources are available to identify specific RBPs responsible for regulating individual AS event. We have developed the OncoSplicing database for integrative analysis of clinically relevant alternative splicing events in TCGA cancers. Here, we further updated the OncoSplicing database by performing correlation analysis between the splicing and mRNA expression data from the TCGA cancers or GTEx tissues, mapping known RNA-binding motifs and eCLIP-seq peaks to all AS events, conducting splicing analysis for RNA-seq data from RBP perturbation experiments in the ENCODE project, and integrating exon and intron sequences for each AS event. With this updated database, users can easily identify potential RBPs responsible for the queried AS event and obtain sequences to design AS-specific primers and minigene constructs for experiment validation. Overall, compared to the previous version, the substantially updated OncoSplicing database (www.oncosplicing.com) offers a more valuable resource for users to identify RBPs responsible for regulating alternative splicing events in cancers.

Participation of retroelements in chromoanagenesis in cancer development

Purpose of the study: to determine the role of retroelements in chromoanagenesis mechanisms in cancer etiopathogenesis.Material and Methods. The search for relevant sources was carried out in the Scopus, Web of Science, PubMed, Elibrary systems, including publications from February 2002 to December 2023. Of the 864 scientifc articles found, 60 were used to write a systematic review.Results. According to original works and meta-analyses results, the cause of complex chromosomal rearrangements during cancer development may be retroelement pathological activation. Chromoanagenesis involves LINE1, SVA, Alu, HERV, which cause double-stranded DNA breaks, insertions in tumor suppressor genes region, the formation of chimeric oncogenes due to retroelement use as new promoters, and function as molecular “band-aids” in non-homologous end junctions and form bridges of distal DNA fragments. Global structural rearrangements of chromosomes observed during chromoanagenesis may be consequences of retroelements activation, which participate in non-allelic homologous recombination and in microhomology-mediated joining of ends characteristic. Certain types of neoplasms, such as colon cancer, are characterized by both high levels of chromothripsis and retroelement activity. In head and neck squamous cell carcinoma, chromoplexy is specifc, the sources of sequences at the breakpoints of which are retroelements. During chromoanagenesis, activation of proto-oncogenes and inactivation of tumor suppressor genes are observed, which is also a consequence of retroelement activation. This is due to the presence of retroelement sequences in proto-oncogenes promoter regions and introns (which become the basis for chimeric oncogene formation) and hot spots of insertional mutagenesis in tumor suppressor genes (transpositions into these regions inactivate these genes).Conclusion. The results obtained on the driver effect of retroelements in chromothripsis, chromoplexy and chromoanasynthesis mechanisms, which are the basis for the formation of clonal evolution of tumors, indicate promise of targeted therapy aimed at silencing the activity of retroelements in cancer patients treatment. For this purpose, it is possible to use microRNAs complementary to retroelements, which are also involved in tumor development, as tools.

RNA binding protein CUGBP2/ETR-3 regulates STAT3 alternative splicing

Signal transducer and activator of transcription 3 (STAT3) is a multifactorial regulator involved in many biological responses. Alternative splicing of STAT3 pre-mRNA leads to an internal 50-nucleotide deletion of exon 23 selecting an alternative 3’ acceptor site, resulting in the generation of two splicing isoforms, STAT3α and STAT3β. STAT3β lacks 55 amino acid-residue transactivation domain at the C-terminal of STAT3α replacing seven unique amino acids. Although STAT3β was originally thought to be a dominant negative isoform of STAT3α, accumulating evidence have shown that STAT3β possesses both its unique functions and those that overlap with STAT3α in fundamental cellular processes. However, much remains unknown about STAT3 pre-mRNA alternative splicing in determining the balance between STAT3 isoforms. In this study, we identified cis-regulatory elements and CUGBP2/ETR-3 as a novel trans-acting factor that regulates STAT3 alternative splicing. Our findings demonstrate that STAT3 splicing can be modulated by CUGBP2 via association with UG-rich elements of intron 22, providing a novel insight into the mechanism of STAT3 alternative splicing. CUGBP2 would be a crucial molecule regulating the balance of STAT3 isoform expression, thus targeting CUGBP2 and its recognition sequences in intron 22 of STAT3 might impact on various biological processes regulated by STAT3 signaling pathway.

Abstract A013: Molecular mechanisms of intron retention in Long Non-Coding RNAs

Abstract Intron retention (IR) is a form of alternative splicing in which an intron that should be spliced out from a precursor transcript, is retained in the mature RNA after the splicing is completed. Although there is emerging evidence of widespread IR in protein-coding genes and long noncoding RNAs (lncRNAs), the underlying molecular mechanisms remain largely unclear. Here, we report the discovery of novel transcripts from the p53-induced lncRNA PURPL, in which intron 2 is retained. To determine the molecular mechanism(s) of IR in PURPL, we conducted a CRISPR-based screen in 3 different cell lines. For this purpose, we utilized a genome-wide guide RNA library expressing a reporter minigene containing the sequence of PURPL intron 2 and its flanking exons. Considering the Percent Intron Retention as readout, we unexpectedly identified proteins of the basal splicing machinery as potential promoting factors of PURPL IR, including the U2-Auxiliary Factor 2 (U2AF2) splicing factor which was one of the top hits of the screen. We next analyzed ENCODE eCLIP-seq datasets to identify RNA binding proteins that could regulate intron 2 and decided to focus on U2AF2 which showed the highest number of binding sites and strongest eCLIP signals within PURPL intron 2 sequence. We validated the effect of U2AF2 by knocking it down, confirming that U2AF2 is a positive regulator of PURPL intron 2 retention as opposed to its canonical function. We also identified the RNA Binding Protein SON as a splicing regulator that acts as an antagonist of U2AF2 in regulating PURPL intron 2 retention. To determine the global impact of U2AF2 knockdown, we performed Iso-Seq and RNA-seq upon U2AF2 depletion. We found that although U2AF2 predominantly acts to promote the splicing of introns in most cellular transcripts, it promotes intron retention in a subset of transcripts. One of these targets is MALAT1, a lncRNA known to play role in splicing by interacting with splicing factors in nuclear speckles. Interestingly, U2AF2 depletion results in MALAT1 exclusion from nuclear speckles. We are currently in the process of characterizing the effect of U2AF2 in the function of the two lncRNAs. These data provide mechanistic insights on PURPL and MALAT1 splicing and function and reveal a previously unrecognized non-canonical function of U2AF2 in promoting intron retention. Citation Format: Ioannis Grammatikakis, Amit Behera, Corrine Corrina R Hartford, Erica C Pehrsson, XiaoLing Li, Yongmei Zhao, Biraj Shrethsa, Tayvia Brownmiller, Natasha J Caplen, Kannanganattu V Prasanth, Thomas Gonatopoulos-Pournatzis, Ashish Lal. Molecular mechanisms of intron retention in Long Non-Coding RNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr A013.

Junk DNA: Is it Really Junk - or a Code Waiting to Be Cracked?

The idea that our genetic material contains Junk DNA is fading. Many DNA segments that were previously believed to be worthless now have important functions, according to recent studies. These non-coding areas, once known as Junk DNA, are crucial for regulating when and how genes function even though they do not directly make proteins. They assist our cells respond to various circumstances and direct the growth of organisms. Certain non-coding DNA sequences function as switches that determine where and when genes should be activated or deactivated. Furthermore, some non-coding DNA segments help maintain the orderly structure of our chromosomes. They also contribute to the evolution of species and may contain remnants of ancient viruses that once infected our forebears. Although scientists still don’t fully understand every aspect of non-coding DNA, it is now recognized as a complex and active element of our biology. Researchers believe that what was once labelled as Junk DNA could actually be very important for our health, development, and ability to adapt to environmental changes. This new perspective is helping us understand how valuable Junk DNA really is.

RNA-First Approach Identifies Deep Intronic PHEX Variants in X-Linked Hypophosphatemic Rickets.

Up to 20% of patients with X-linked hypophosphatemic rickets (XLH) have no causative variant identified on routine molecular diagnostic testing. To identify intronic variants causing PHEX mis-splicing in patients with XLH. The metabolic bone clinic of a paediatric orthopedic hospital. Four patients (age 6 to 12 years; 3 girls) with clinically diagnosed XLH and no PHEX variant on routine testing. RNA and DNA sequence analysis of PHEX. Urine-derived cells were cultured, and mRNA was extracted and transcribed to cDNA. PHEX cDNA was amplified by PCR, followed by sequencing of PCR products. Sequencing of PHEX intronic DNA regions was performed to identify variants causing mis-splicing observed on RNA analysis. PHEX mis-splicing was identified in 3 of the 4 participants, and an intronic variant was identified in all 3 cases. In a 12-year-old boy, transcript analysis showed skipping of PHEX exon 13, while sequencing of PHEX intronic regions revealed a de novo 18 bp deletion in intron 13. In a 7-year-old girl, a pseudoexon in PHEX intron 17 was found, associated with a de novo deep intronic variant (c.1768+173A>G) that activated a cryptic splice donor site. Finally, an 84 bp pseudoexon in PHEX intron 21 caused by a recurrent de novo deep intronic variant (c.2147+1197A>G) was identified in an 11-year-old girl. Analysis of RNA from urine-derived cells combined with sequencing of PHEX introns can identify deep intronic variants in individuals with XLH without a detectable PHEX variant in routine exon-centric molecular diagnosis.

Single-cell eQTL mapping in yeast reveals a tradeoff between growth and reproduction.

Expression quantitative trait loci (eQTLs) provide a key bridge between noncoding DNA sequence variants and organismal traits. The effects of eQTLs can differ among tissues, cell types, and cellular states, but these differences are obscured by gene expression measurements in bulk populations. We developed a one-pot approach to map eQTLs in Saccharomyces cerevisiae by single-cell RNA sequencing (scRNA-seq) and applied it to over 100,000 single cells from three crosses. We used scRNA-seq data to genotype each cell, measure gene expression, and classify the cells by cell-cycle stage. We mapped thousands of local and distant eQTLs and identified interactions between eQTL effects and cell-cycle stages. We took advantage of single-cell expression information to identify hundreds of genes with allele-specific effects on expression noise. We used cell-cycle stage classification to map 20 loci that influence cell-cycle progression. One of these loci influenced the expression of genes involved in the mating response. We showed that the effects of this locus arise from a common variant (W82R) in the gene GPA1 , which encodes a signaling protein that negatively regulates the mating pathway. The 82R allele increases mating efficiency at the cost of slower cell-cycle progression and is associated with a higher rate of outcrossing in nature. Our results provide a more granular picture of the effects of genetic variants on gene expression and downstream traits.

Analysis of the variations within Q. ilex L. and the evaluation of morphological types based on chloroplast and nuclear DNA sequences

Q. ilex evaluated within evergreen has wide geographic distribution in Mediterranean basin. Hybridization and gene flow are effective and frequently observed mechanisms in Q. ilex. Moreover, weak reproductive barrier between closely related taxa in the zones with geographical contact is another important state that increase genetic diversity within Q. ilex and then taxonomic problems. Two morphological types known as rotundifolia and ilex are defined based on the variations between Q. ilex populations appearing as a result of all these factors. However, it is still controversial situation whether morphological types: ilex and rotundifolia are subspecies of Q. ilex or two separate species. In this study, short DNA sequences that consist of matK gene-partial trnK gene intron of chloroplast DNA and ITS1-5.8S rRNA gene-ITS2 of nuclear DNA were used to overcome such difficulties and to reveal the variations between Q. ilex populations. All populations belonging to Q. ilex based on both barcoding regions were determined and examined using the Molecular Evolutionary Genetics Analysis (MEGA 11). The analysis such as base substitutions, variable and parsim-info sites, transitional and transversional base substitution ranges (%) and nucleotide frequencies (%) were performed and finally, Maximum Parsimony (MP) dendrograms for both barcoding regions were drawn to evaluate the populations belonging to Q. ilex in terms of their variations, phylogenetic-evolutionary relationships and taxonomic statues. Although both barcoding regions support the separation of Q. ilex populations based on different morphological types, especially, matK gene-partial trnK gene intron sequences exhibited more clear and informative results than ITS1-5.8S rRNA gene-ITS2 sequences.

Hybrid Origin of ×Leymotrigia bergrothii (Poaceae) as Revealed by Analysis of the Internal Transcribed Spacer ITS1 and trnL Sequences.

×Leymotrigia bergrothii is a presumed hybrid of Leymus arenarius and Elytrigia repens. This article investigates the hybrid origin and genome composition of this species. These plants are sterile, do not undergo pollination, and do not produce seeds; occasionally, underdeveloped stamens containing abortive pollen grains form in individual spikelets. The karyotype analysis of root meristem cells revealed a diploid chromosome number of 49 in ×L. bergrothii, reported here for the first time. Subsequently, we examined the intragenomic polymorphism of the transcribed spacer ITS1 in several species of Elytrigia, Elymus, Leymus, Hordeum, and Psathyrostachys, and compared the ribotype patterns of these species with those of ×L. bergrothii. It is shown that the St-ribotype variants found in Elytrigia repens and Elytrigia pseudocaesia, as well as the ribotypes of the La family, which dominate in the genome of Leymus arenarius, correspond to major ribotypes in ×L. bergrothii. The ribotypes of the St and La families are present in the nuclear genome of ×L. bergrothii in almost equal proportions. A comparison of intron and exon sequences of the trnL gene in the chloroplast DNA of Leymus arenarius, Elytrigia repens, and ×L. bergrothii showed that this region in ×L. bergrothii is identical or very close to that of Elytrigia repens, suggesting that Elytrigia repens was the cytoplasmic donor to ×L. bergrothii. Thus, our study confirms the hypothesis that this species represents a sterile first-generation hybrid of Leymus arenarius and Elytrigia repens, reproducing vegetatively.

The first chicken oocyte nucleus whole transcriptomic profile defines the spectrum of maternal mRNA and non-coding RNA genes transcribed by the lampbrush chromosomes.

Lampbrush chromosomes, with their unusually high rate of nascent RNA synthesis, provide a valuable model for studying mechanisms of global transcriptome up-regulation. Here, we obtained a whole-genomic profile of transcription along the entire length of all lampbrush chromosomes in the chicken karyotype. With nuclear RNA-seq, we obtained information about a wider set of transcripts, including long non-coding RNAs retained in the nucleus and stable intronic sequence RNAs. For a number of protein-coding genes, we visualized their nascent transcripts on the lateral loops of lampbrush chromosomes by RNA-FISH. The set of genes transcribed on the lampbrush chromosomes is required for basic cellular processes and is characterized by a broad expression pattern. We also present the first high-throughput transcriptome characterization of miRNAs and piRNAs in chicken oocytes at the lampbrush chromosome stage. Major targets of predicted piRNAs include CR1 and long terminal repeat (LTR) containing retrotransposable elements. Transcription of tandem repeat arrays was demonstrated by alignment against the whole telomere-to-telomere chromosome assemblies. We show that transcription of telomere-derived RNAs is initiated at adjacent LTR elements. We conclude that hypertranscription on the lateral loops of giant lampbrush chromosomes is required for synthesizing large amounts of transferred to the embryo maternal RNA for thousands of genes.

Expression Profiling Analysis of the SWEET Gene Family in In Vitro Pitaya Under Low-Temperature Stress and Study of Its Cold Resistance Mechanism.

Pitaya (Hylocereus undatus) fruit is an attractive, nutrient-rich tropical fruit with commercial value. However, low-temperature stress severely affects the yield and quality of pitaya. The relevant mechanisms involved in the response of pitaya to low-temperature stress remain unclear. To study whether the SWEET gene family mediates the response of H. undatus to low-temperature stress and the related mechanisms, we performed genome-wide identification of the SWEET gene family in pitaya, and we used 'Baiyulong' tissue-cultured plantlets as material in the present study. We identified 28 members of the SWEET gene family from the H. undatus genome and divided these family members into four groups. Members of this gene family presented some differences in the sequences of introns and exons, but the gene structure, especially the motifs, presented relatively conserved characteristics. The promoter regions of most HuSWEETs have multiple stress- or hormone-related cis-elements. Three duplicated gene pairs were identified, including one tandem duplication gene and two fragment duplication gene pairs. The results revealed that the SWEET genes may regulate the transport and distribution of soluble sugars in plants; indirectly regulate the enzyme activities of CAT, POD, and T-SOD through its expression products; and are involved in the response of pitaya to low-temperature stress and play vital roles in this process. After ABA and MeJA treatment, the expression of HuSWEETs changed significantly, and the cold stress was also alleviated. This study elucidated the molecular mechanism and physiological changes in the SWEET gene in sugar metabolism and distribution of pitaya when it experiences low-temperature stress and provided a theoretical basis for cold-resistant pitaya variety breeding.

Genetic and Epigenetic Dysregulation of Transcriptional Enhancers in Cancer

Enhancers are noncoding DNA sequences responsible for orchestrating gene expression programs by interacting with transcription factors and chromatin regulators within complex genome structures. However, their fundamental functions can be disrupted by genetic and epigenetic alterations, leading to aberrant enhancer activation or rewiring that contributes to oncogenesis. Analyzing dysregulated enhancer landscapes reveals new subtype-defining genomic features, such as enhancer hijacking, and identifies disease-relevant transcriptional regulators as potential targets for developing enhancer-targeting therapeutic strategies. Here, we delve into evolving concepts and technologies for studying enhancers; discuss how genetic, epigenetic, and topological alterations disrupt enhancer regulation to promote oncogenic gene expression; and underscore the importance of understanding the regulatory principles governing enhancer function to guide therapeutic strategies. Furthermore, we highlight key challenges and emerging opportunities in targeting enhancers and their regulators to improve cancer classification, prognosis, and treatment.

Intronic regulatory sequences regulate epicardial gene expression and EMT during heart development

Abstract Background During organogenesis, cells from the outer layer of the heart, the epicardium, undergo epithelial-to-mesenchymal transition (EMT), contributing essential paracrine signals and different cell types to the growing heart. Epicardial cells are integral to heart regeneration in lower vertebrates and neonatal mammalian injured hearts. That said, prospects to harness the epicardium for therapeutic applications to effect adult heart repair, heavily depend on improved insight into its intrinsic properties during heart development. Purpose Cell fate decisions underpinning EMT are directed by transcription factors such as Wilms’ tumour 1 (WT1). Whilst a requirement for Wt1 in heart development is widely accepted, the upstream regulatory mechanisms underpinning its activation remain elusive. Our previous research has led to the identification of two intronic evolutionary conserved regions (ECRs) shared between mouse and human, located within intron 1 of the Wt1 locus, i.e. +4.0kbECR and +5.8kbECR. We hypothesised these regulatory sequences direct locus activation and EMT to support normal heart development. Methods & Results Here, we used CRISPR/Cas9 gene-editing technology to generate mice carrying a sequence deletion containing one ECR (Wt1Δ+4.0kbECR and Wt1Δ+5.8kbECR mice) or a deletion comprising both ECRs (Wt1Δ+4-5.8kbECR mouse). Extensive survival analysis, high-resolution episcopic microscopy (HREM), qPCR, immunostaining, confocal microscopy and epicardial explants were used to characterise heart formation in these models. Mendelian ratios indicated an overall underrepresentation of Wt1ΔECR/ΔECR mutants recovered in the offspring arising from heterozygous inter-crosses. HREM revealed smaller hearts, incidence of myocardial non-compaction and spongy interventricular septum with muscular or membranous ventricular septum defects, tricuspid hypoplasia and enlarged aortic valves, as well as abnormal formation/patterning of coronary artery stems in ΔECR/ΔECR hearts. Expression of Wt1 was markedly reduced in ΔECR/ΔECR hearts, but not in the kidneys, suggesting intronic enhancers are cardiac-specific. Whole-mount and tissue-section immunostaining combined with confocal microscopy revealed abnormal coronary vessel and innervation extension and patterning along the subepicardial space, as well as reduced epicardial EMT and myocardial non-compaction/hyper-trabeculation in Wt1ΔECR/ΔECR hearts. Conclusions Together, we demonstrated a requirement for novel intronic enhancers regulating Wt1 locus activity and impacting on essential epicardial EMT and associated biological processes during normal heart development. Importantly, observation of septum and aortic valve defects in ΔECR mutants suggests an hitherto unrecognised role for WT1/epicardial EMT and opens new avenues of research to improve our understanding of congenital heart disease that affects at least 1:150 live births, with remarkably two thirds of cases having unknown aetiology.

Epithelial splicing regulatory protein 1 promotes peritoneal dissemination of ovarian cancer by inducing the formation of circular RNAs modulating epithelial plasticity

Peritoneal metastases prevalently occur in ovarian cancer, deteriorating patient prognosis. During the metastatic cascade, tumor plasticity enables cells to adapt to environmental changes, thereby facilitating dissemination. We previously found that epithelial splicing regulatory protein 1 (ESRP1) is linked to peritoneal metastasis and epithelial-mesenchymal plasticity in ovarian cancer. This study delves into the underlying mechanism. We found that ESRP1 preserves epithelial plasticity in ovarian cancer cells in vitro and in vivo. Functionally, ESRP1 enhances ovarian cancer cell growth and peritoneal dissemination. High-throughput sequencing revealed several ESRP1-related epithelial RNAs, encompassing both linear and circular forms. Specifically, ESRP1 triggers the cyclization of circPAFAH1B2 and circUBAP2 through binding to the GGU sequences in adjacent introns. The two ESRP1-induced circular RNAs stabilize DKK3 and AHR mRNAs, which are critical for epithelial plasticity, through interaction with IGF2BP2. Collectively, ESRP1 triggers the formation of circPAFAH1B2 and circUBAP2, which in turn stabilizes DKK3 and AHR through IGF2BP2 binding, thereby modulating the epithelial plasticity and aiding the peritoneal spread of ovarian cancer cells. The findings unveiled a biological network, orchestrated by ESRP1, that governs the epithelial-mesenchymal plasticity of ovarian cancer cells, emphasizing the therapeutic potential of ESRP1 and its induced circular RNAs for ovarian cancer treatment.

Chloroplast genome sequencing and divergence analysis of 18 Pyrus species: insights into intron length polymorphisms and evolutionary processes.

Pears constitute an essential temperate crop and are primarily produced through interspecific hybridization owing to self-incompatibility that complicates their breeding history. To address this, we sequenced the complete chloroplast (cp) genomes of 18 Pyrus and one Malus species using the Illumina HiSeq4000 platform. The cp genomes ranged from 159,885 bp to 160,153bp and exhibited a conserved circular DNA structure with an average GC content of 36.5%. Each cp genome contained 127 genes, including 83 protein-coding, 36 tRNA, and 8 rRNA genes. Divergence analysis with mVISTA showed high conservation in the coding regions and notable variations in the non-coding regions. All species shared 17 intron-containing genes, with ycf3 and clpP each having two introns. Five intron-containing genes (ndhB, rpl2, rps12, trnA-UGC, and trnE-UUC) were located in the inverted repeat regions, while trnL-UAA was located in the large single-copy region, with conserved intron lengths across Pomoideae. We identified polymorphic intron sequences in the rpl22, petB, clpP, ndhA, and rps16 genes and designed primers for these regions. Notably, the two Pyrus ussuriensis accessions Doonggeullebae and Cheongdangrori showed intron-length polymorphisms despite being classified as the same species. Phylogenetic analysis of the cp genome sequences revealed two major clusters, indicating distinct maternal lineages and evolutionary origins. This study underscores the importance of cp gene polymorphisms in P. fauriei, P. calleryana, P. ussuriensis, and P. pyrifolia, providing valuable insights into Pyrus evolution as well as aiding in the conservation and breeding of pear germplasm.

Global nuclear reorganization during heterochromatin replication in the giant-genome plant Nigella damascena L.

Among flowering plants, genome size varies remarkably, by >2200-fold, and this variation depends on the loss and gain of noncoding DNA sequences that form distinct heterochromatin complexes during interphase. In plants with giant genomes, most chromatin remains condensed during interphase, forming a dense network of heterochromatin threads called interphase chromonemata. Using super-resolution light and electron microscopy, we studied the ultrastructure of chromonemata during and after replication in root meristem nuclei of Nigella damascena L. During S-phase, heterochromatin undergoes transient decondensation locally at DNA replication sites. Due to the abundance of heterochromatin, the replication leads to a robust disassembly of the chromonema meshwork and a general reorganization of the nuclear morphology visible even by conventional light microscopy. After replication, heterochromatin recondenses, restoring the chromonema structure. Thus, we show that heterochromatin replication in interphase nuclei of giant-genome plants induces a global nuclear reorganization.

DOCK8 deficiency due to a deep intronic variant in two kindreds with hyper-IgE syndrome

Dedicator of cytokinesis 8 (DOCK8) deficiency underlies the majority of cases of patients with autosomal recessive form of the hyper-immunoglobulin E syndrome (HIES). Most DOCK8 mutations involve deletions and splice junction mutations that abrogate protein expression. However, a few patients whose presentation is reminiscent of DOCK8 deficiency have no identifiable mutations. Using Whole Exome Sequencing (WES), we identified a deep intronic homozygous DOCK8 variant located in intron 36 (c.4626 + 76 A > G) in two unrelated patients with features of HIES that resulted in an in-frame 75 base pair intronic sequence insertion in DOCK8 cDNA, resulting in a premature stop codon (p.S1542ins6Ter). This variant resulted in variable decrease in DOCK8 expression that was associated with impaired T cell receptor-triggered actin polymerization, decreased IL-6-induced STAT3 phosphorylation, reduced expression of the Th17 cell markers CCR6 and IL-17, and higher frequencies of GATA3+ T cells indicative of Th2 skewing. Our approach extends the reach of WES in identifying disease-related intronic variants. It highlights the role of non-coding mutations in immunodeficiency disorders, including DOCK8 deficiency, and emphasizes the need to explore these mutations in unexplained inborn errors of immunity.

Widespread HCD-tRNA derived SINEs in bivalves rely on multiple LINE partners and accumulate in genic regions

BackgroundShort interspersed nuclear elements (SINEs) are non-autonomous non-LTR retrotransposons widespread across eukaryotes. They exist both as lineage-specific, fast-evolving elements and as ubiquitous superfamilies characterized by highly conserved domains (HCD). Several of these superfamilies have been described in bivalves, however their overall distribution and impact on host genome evolution are still unknown due to the extreme scarcity of transposon libraries for the clade. In this study, we examined more than 40 bivalve genomes to uncover the distribution of HCD-tRNA-related SINEs, discover novel SINE-LINE partnerships, and understand their possible role in shaping bivalve genome evolution.ResultsWe found that bivalve HCD SINEs have an ancient origin, and they can rely on at least four different LINE clades. According to a “mosaic” evolutionary scenario, multiple LINE partner can promote the amplification of the same HCD SINE superfamilies while homologues LINE-derived tails are present between different superfamilies. Multiple SINEs were found to be highly similar between phylogenetically related species but separated by extremely long evolutionary timescales, up to ~ 400 million years. Studying their genomic distribution in a subset of five species, we observed different patterns of SINE enrichment in various genomic compartments as well as differences in the tendency of SINEs to form tandem-like and palindromic structures also within intronic sequences. Despite these differences, we observed that SINEs, especially older ones, tend to accumulate preferentially within genes, or in their close proximity, consistently with a model of survival bias for less harmful, short non-coding transposons in euchromatic genomic regions.ConclusionHere we conducted a wide characterization of tRNA-related SINEs in bivalves revealing their taxonomic distribution and LINE partnerships across the clade. Moreover, through the study of their genomic distribution in five species, we highlighted commonalities and differences with other previously studied eukaryotes, thus extending our understanding of SINE evolution across the tree of life.

A Comprehensive Analysis of CSN1S2 I and II Transcripts Reveals Significant Genetic Diversity and Allele-Specific Exon Skipping in Ragusana and Amiatina Donkeys.

The αs2-casein is a phosphoprotein secreted in the milk of most mammals, and it is the most hydrophilic of all caseins. Contrary to genes found in ruminants, in donkeys two different encoding genes for donkey αs2-casein (CSN1S2 I and CSN1S2 II) have been identified. However, unlike in ruminants, the variability at these loci has not been characterized in detail in donkeys until now. In this study, we analyze the transcript profile of the donkey CSN1S2 I and CSN1S2 II genes, and we identify and describe the variability of these loci in the Ragusana and Amiatina breeds reared in Italy. The analysis of the CSN1S2 I Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) products and subsequent sequencing showed, in addition to correctly spliced mRNA, seven other minor mRNAs resulting from differential splicing events involving, in various combinations, entire exons (4, 5, 6, and 11), parts of exons (5' or 3' end of exon 17), or the recognition of intronic sequences as an exon (exon 12'). Similarly, the transcription analysis of the CSN1S2 II gene revealed a remarkable variability in splicing events, mainly concerning the alternative insertion of an extra exon 7 (named 7'); the first 33 bp of exon 13; or the alternative skipping of exons 9, 10, 11, 12, and 15, and their combinations. At the mRNA level for CSN1S2 I, seven SNPs were observed, five of which led to amino acid changes: p.T73>A, p.I109>V, p.I130>V, p.I146>T, and p.D217>Y. Similarly, nine SNPs were observed at the CSN1S2 II locus, seven of which are non-synonymous: p.L63>F, p.H70>Q, p.D90>N, p.129A>T, p.H131>Y, p.E144>G, and p.F157>S. In addition, the DNA sequencing of exon 17 and flanking introns of the CSN1S2 I gene revealed a G>A transition at the splice acceptor site of CSN1S2 I exon 17 (FM946022.1:c.375-1G>A), resulting in an allele-specific skipping of the first 15 nucleotides of this exon, which encode the peptide 176NKINQ180, and the recognition of an in-frame cryptic splicing acceptor site: arAACAAAATCAACCAG. A genotyping method based on restriction fragment length polymorphism (XbaI PCR-RFLP) was set up for this SNP. In the total population studied (105 Ragusana and 14 Amiatina donkeys), the A allele had a frequency of 0.2437 with no evidence of deviation from the Hardy-Weinberg equilibrium. This study adds new knowledge regarding the genetic variability of αs2-caseins in donkeys and may contribute significantly to the genetic improvement of milk production for this species.