Search

Background: It is generally accepted that nuclear genes in eukaryotes are located independently on chromosomes and expressed in a monocistronic manner. However, accumulating evidence suggests a more complex landscape of gene structure and transcription. Ganoderma lingzhi, a model medicinal fungus, currently lacks high-quality genome annotation, hindering genetic studies. Results: Here, we reported a golden annotation of G. lingzhi, featuring 14,147 high-confidence genes derived from extensive manual corrections. Novel characteristics of gene structure and transcription were identified accordingly. Notably, non-canonical splicing sites accounted for 1.99% of the whole genome, with the predominant types being GC-AG (1.85%), GT-AC (0.05%), and GT-GG (0.04%). 1165 pairs of genes were found to have overlapped transcribed regions, and 92.19% of which showed opposite directions of gene transcription. A total of 5,412,158 genetic variations were identified among 13 G. lingzhi strains, and the manually corrected gene sets resulted in enhanced functional annotation of these variations. More than 60% of G. lingzhi genes were alternatively spliced. In addition, we found that two or more protein-coding genes (PCGs) can be transcribed into a single RNA molecule, referred to as polycistronic genes. In total, 1272 polycistronic genes associated with 2815 PCGs were identified. Conclusions: The widespread presence of polycistronic genes in G. lingzhi strongly complements the theory that polycistron is also present in eukaryotic genomes. The extraordinary gene structure and transcriptional activity uncovered through this golden annotation provide implications for the study of genes, genomes, and related studies in G. lingzhi and other eukaryotes. [ABSTRACT FROM AUTHOR]

Deep learning models (DLMs) have gained importance in predicting, detecting, translating, and classifying a diversity of inputs. In bioinformatics, DLMs have been used to predict protein structures, transcription factor-binding sites, and promoters. In this work, we propose a hybrid model to identify transcription factors (TFs) among prokaryotic and eukaryotic protein sequences, named Deep Regulation (DeepReg) model. Two architectures were used in the DL model: a convolutional neural network (CNN), and a bidirectional long-short-term memory (BiLSTM). DeepReg reached a precision of 0.99, a recall of 0.97, and an F1-score of 0.98. The quality of our predictions, the bias-variance trade-off approach, and the characterization of new TF predictions were evaluated and compared against those produced by DeepTFactor, as well as against experimental data from three model organisms. Predictions based on our DLM tended to exhibit less variance and bias than those from DeepTFactor, thus increasing reliability and decreasing overfitting. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are mobile genomic elements constituting a big fraction of eukaryotic genomes. They ignite an evolutionary arms race with host genomes, which in turn evolve strategies to restrict their activity. Despite being tightly repressed, TEs display precisely regulated expression patterns during specific stages of mammalian development, suggesting potential benefits for the host. Among TEs, the long interspersed nuclear element (LINE‐1 or L1) has been found to be active in neurons. This activity prompted extensive research into its possible role in cognition. So far, no specific cause‐effect relationship between L1 retrotransposition and brain functions has been conclusively identified. Nevertheless, accumulating evidence suggests that interactions between L1 RNAs and RNA/DNA binding proteins encode specific messages that cells utilize to activate or repress entire transcriptional programs. We summarize recent findings highlighting the activity of L1 RNAs at the non‐coding level during early embryonic and brain development. We propose a hypothesis suggesting a mutualistic relationship between L1 mRNAs and the host cell. In this scenario, cells tolerate a certain rate of retrotransposition to leverage the regulatory effects of L1s as non‐coding RNAs on potentiating their mitotic potential. In turn, L1s benefit from the cell's proliferative state to increase their chance to mobilize. [ABSTRACT FROM AUTHOR]

Guanine-rich DNA forms G-quadruplexes (G4s) that play a critical role in essential cellular processes. Previous studies have mostly focused on intramolecular G4s composed of four consecutive guanine tracts (G-tracts) from a single strand. However, this structural form has not been strictly confirmed in the genome of living eukaryotic cells. Here, we report the formation of hybrid G4s (hG4s), consisting of G-tracts from both DNA and RNA, in the genome of living yeast cells. Analysis of Okazaki fragment syntheses and two other independent G4-specific detections reveal that hG4s can efficiently form with as few as a single DNA guanine-guanine (GG) tract due to the participation of G-tracts from RNA. This finding increases the number of potential G4-forming sites in the yeast genome from 38 to 587,694, a more than 15,000-fold increase. Interestingly, hG4s readily form and even dominate at G4 sites that are theoretically capable of forming the intramolecular DNA G4s (dG4s) by themselves. Compared to dG4s, hG4s exhibit broader kinetics, higher prevalence, and greater structural diversity and stability. Most importantly, hG4 formation is tightly coupled to transcription through the involvement of RNA, allowing it to function in a transcription-dependent manner. Overall, our study establishes hG4s as the overwhelmingly dominant G4 species in the yeast genome and emphasizes a renewal of the current perception of the structural form, formation mechanism, prevalence, and functional role of G4s in eukaryotic genomes. It also establishes a sensitive and currently the only method for detecting the structural form of G4s in living cells. [ABSTRACT FROM AUTHOR]

CRISPR-associated (Cas) endonucleases and their derivatives are widespread tools for the targeted genetic modification of both prokaryotic and eukaryotic genomes. A critical step of all CRISPR-Cas technologies is the delivery of the Cas endonuclease to the target cell. Here, we investigate the possibility of using bacterial conjugation to translocate Cas proteins into recipient bacteria. Conjugative relaxases are translocated through a type IV secretion system into the recipient cell, covalently attached to the transferred DNA strand. We fused relaxase R388-TrwC with the endonuclease Cas12a and confirmed that it can be transported through a T4SS. The fusion protein maintained its activity upon translocation by conjugation into the recipient cell, as evidenced by the induction of the SOS signal resulting from DNA breaks produced by the endonuclease in the recipient cell, and the detection of mutations at the target position. We further show how a template DNA provided on the transferred DNA can be used to introduce specific mutations. The guide RNA can also be encoded by the transferred DNA, enabling its production in the recipient cells where it can form a complex with the Cas nuclease transferred as a protein. This self-contained setup enables to target wild-type bacterial cells. Finally, we extended this strategy to the delivery of relaxases fused to base editors. Using TrwC and MobA relaxases as drivers, we achieved precise editing of transconjugants. Thus, conjugation provides a delivery system for Cas-derived editing tools, bypassing the need to deliver and express a cas gene in the target cells. [ABSTRACT FROM AUTHOR]

Summary: Horizontal gene transfer (HGT) is a major driving force in the evolution of prokaryotic and eukaryotic genomes. Despite recent advances in distribution and ecological importance, the extensive pattern, especially in seed plants, and post‐transfer adaptation of HGT‐acquired genes in land plants remain elusive.We systematically identified 1150 foreign genes in 522 land plant genomes that were likely acquired via at least 322 distinct transfers from nonplant donors and confirmed that recent HGT events were unevenly distributed between seedless and seed plants.HGT‐acquired genes evolved to be more similar to native genes in terms of average intron length due to intron gains, and HGT‐acquired genes containing introns exhibited higher expression levels than those lacking introns, suggesting that intron gains may be involved in the post‐transfer adaptation of HGT in land plants.Functional validation of bacteria‐derived gene GuaD in mosses and gymnosperms revealed that the invasion of foreign genes introduced a novel bypass of guanine degradation and resulted in the loss of native pathway genes in some gymnosperms, eventually shaping three major types of guanine metabolism in land plants. We conclude that HGT has played a critical role in land plant evolution. [ABSTRACT FROM AUTHOR]

ABSTRACT Eukaryotic genomes harbour sequences derived from non‐retroviral RNA viruses, known as endogenous viral elements (EVEs) or non‐retroviral integrated RNA virus sequences (NIRVS). These sequences represent a record of past infections and have been implicated in host anti‐viral response. We have created a program to identify viral sequences integrated in a host genome. It begins with a specimen BAM file and outputs candidate NIRVS, along with putative host insertion sites and overlapping genomic features of the host genome in XML and visual formats, with minimal intermediary intervention. We ran through this software short‐read data derived from the genomes of 222 wild‐caught A. aegypti mosquitoes, from a dozen geographical regions, and located putative NIRVS from seven virus families. This program is as accurate as currently available software for NIRVS detection, and represents a significant improvement in adaptability and user‐friendliness. Furthermore, the flexibility of this pipeline allows the user to search for sequence integrations across the genome of any organism, as long as a query sequence database and a reference genome is provided. Potential extended applications include identification of integrated transgenic sequences used for research or vector control strategies. [ABSTRACT FROM AUTHOR]

Repeating sequences of DNA, or repetitive elements (REs), are common features across both prokaryotic and eukaryotic genomes. Unlike many of their protein-coding counterparts, the functions of REs in host cells remained largely unknown and have often been overlooked. While there is still more to learn about their functions, REs are now recognized to play significant roles in both beneficial and pathological processes in their hosts at the cellular and organismal levels. Therefore, in this review, we discuss the various types of REs and review what is known about their evolution. In addition, we aim to classify general mechanisms by which REs promote processes that are variously beneficial and harmful to host cells/organisms. Finally, we address the emerging role of REs in cancer, aging, and neurological disorders and provide insights into how RE modulation could provide new therapeutic benefits for these specific conditions. [ABSTRACT FROM AUTHOR]

Human APOBEC single-strand (ss) specific DNA and RNA cytidine deaminases change cytosines to uracils (U's) and function in antiviral innate immunity and RNA editing and can cause hypermutation in chromosomes. The resulting U's can be directly replicated, resulting in C to T mutations, or U–DNA glycosylase can convert the U's to abasic (AP) sites which are then fixed as C to T or C to G mutations by translesion DNA polymerases. We noticed that in yeast and in human cancers, contributions of C to T and C to G mutations depend on the origin of ssDNA mutagenized by APOBECs. Since ssDNA in eukaryotic genomes readily binds to replication protein A (RPA) we asked if RPA could affect APOBEC-induced mutation spectrum in yeast. For that purpose, we expressed human APOBECs in the wild-type (WT) yeast and in strains carrying a hypomorph mutation rfa1-t33 in the large RPA subunit. We confirmed that the rfa1-t33 allele can facilitate mutagenesis by APOBECs. We also found that the rfa1-t33 mutation changed the ratio of APOBEC3A-induced T to C and T to G mutations in replicating yeast to resemble a ratio observed in long persistent ssDNA in yeast and in cancers. We present the data suggesting that RPA may shield APOBEC formed U's in ssDNA from Ung1, thereby facilitating C to T mutagenesis through the accurate copying of U's by replicative DNA polymerases. Unexpectedly, we also found that for U's shielded from Ung1 by WT RPA, the mutagenic outcome is reduced in the presence of translesion DNA polymerase zeta. [ABSTRACT FROM AUTHOR]

Summary: Synthetic genomics involves the design, assembly, and transfer of artificially synthesized DNA fragments into target hosts to replace the native genome and construct viable forms of life. With advances in DNA synthesis and assembly techniques, the application of synthetic genomics in viruses, bacteria, and yeast has improved our knowledge of genome organization and function. Multicellular eukaryotic organisms are characterized by larger genomes, more complex epigenetic regulation, and widespread transposable elements, making genome synthesis challenging. Recently, the first synthetic multicellular eukaryotic organism was generated in the model plant Physcomitrium patens with a partially synthetic chromosome arm. Here, we introduce the design and assembly principles of moss genome synthesis. We also discuss the remaining technical barriers in the application of synthetic genomics in seed plants. [ABSTRACT FROM AUTHOR]

ABSTRACT DNA in eukaryotic genomes is under constant assault from both exogenous and endogenous sources, leading to DNA damage, which is considered a major molecular driver of ageing. Fortunately, the genome and the central exome are safeguarded against these attacks by abundant peripheral non‐coding DNA. Non‐coding DNA codes for small non‐coding RNAs that inactivate foreign nucleic acids in the cytoplasm and physically blocks these attacks in the nucleus. Damage to non‐coding DNA produced during such blockage is removed in the form of extrachromosomal circular DNA (eccDNA) through nucleic pore complexes. Consequently, non‐coding DNA serves as a line of defence for the exome against DNA damage. The total amount of non‐coding DNA/heterochromatin declines with age, resulting in a decrease in both physical blockage and eccDNA exclusion, and thus an increase in the accumulation of DNA damage in the nucleus during ageing and in age‐related diseases. Here, we summarize recent evidence supporting a protective role of non‐coding DNA in healthy and pathological states and argue that DNA damage is the proximate cause of ageing and age‐related genetic diseases. Strategies aimed at strengthening the protective role of non‐coding DNA/heterochromatin could potentially offer better systematic protection for the dynamic genome and the exome against diverse assaults, reduce the burden of DNA damage to the exome, and thus slow ageing, counteract age‐related genetic diseases and promote a healthier life for individuals. [ABSTRACT FROM AUTHOR]

Background: The WD40 domain, one of the most abundant in eukaryotic genomes, is widely involved in plant growth and development, secondary metabolic biosynthesis, and mediating responses to biotic and abiotic stresses. WD40 repeat (WD40) protein has been systematically studied in several model plants but has not been reported in the Capsicum annuum (pepper) genome. Results: Herein, 269, 237, and 257 CaWD40 genes were identified in the Zunla, CM334, and Zhangshugang genomes, respectively. CaWD40 sequences from the Zunla genome were selected for subsequent analysis, including chromosomal localization, phylogenetic relationships, sequence characteristics, motif compositions, and expression profiling. CaWD40 proteins were unevenly distributed on 12 chromosomes, encompassing 19 tandem duplicate gene pairs. The 269 CaWD40s were divided into six main branches (A to F) with 17 different types of domain distribution. The CaWD40 gene family exhibited diverse expression patterns, and several genes were specifically expressed in flowers and seeds. Yeast two-hybrid (Y2H) and dual-luciferase assay indicated that CaWD40-91 could interact with CaAN1 and CaDYT1, suggesting its involvement in anthocyanin biosynthesis and male sterility in pepper. Conclusions: In summary, we systematically characterized the phylogeny, classification, structure, and expression of the CaWD40 gene family in pepper. Our findings provide a valuable foundation for further functional investigations on WD40 genes in pepper. [ABSTRACT FROM AUTHOR]

Transcription of antisense long noncoding RNAs (lncRNAs) occurs pervasively across eukaryotic genomes. Only a few antisense lncRNAs have been characterized and shown to control biological processes, albeit with idiosyncratic regulatory mechanisms. Thus, we largely lack knowledge about the general role of antisense transcription in eukaryotic organisms. Here, we characterized genes with antisense transcription initiating close to the poly(A) signal of genes (PAS genes) in Arabidopsis (Arabidopsis thaliana). We compared plant native elongation transcript sequencing (plaNET-seq) with RNA sequencing during short-term cold exposure and detected massive differences between the response in active transcription and steady-state levels of PAS gene-derived mRNAs. The cold-induced expression of transcription factors B-BOX DOMAIN PROTEIN28 (BBX28) and C2H2-TYPE ZINC FINGER FAMILY PROTEIN5 (ZAT5) was detected by plaNET-seq, while their steady-state level was only slightly altered due to high mRNA turnover. Knockdown of BBX28 and ZAT5 or of their respective antisense transcripts severely compromised plant freezing tolerance. Decreased antisense transcript expression levels resulted in a reduced cold response of BBX28 and ZAT5, revealing a positive regulatory role of both antisense transcripts. This study expands the known repertoire of noncoding transcripts. It highlights that native transcription approaches can complement steady-state RNA techniques to identify biologically relevant players in stress responses. Genes that host antisense transcripts are often stress-responsive and important for plant stress acclimation, even though their mRNA steady-state level is only marginally altered by stress. [ABSTRACT FROM AUTHOR]

Summary Identification of allelic or corresponding genes (pan-genes) within a species or genus is important for discovery of biologically significant genetic conservation and variation. Similarly, identification of orthologs (gene families) across wider evolutionary distances is important for understanding the genetic basis for similar or differing traits. Especially in plants, several complications make identification of pan-genes and gene families challenging, including whole-genome duplications, evolutionary rate differences among lineages, and varying qualities of assemblies and annotations. Here, we document and distribute a set of workflows that we have used to address these problems. Results Pandagma is a set of configurable workflows for identifying and comparing pan-gene sets and gene families for annotation sets from eukaryotic genomes, using a combination of homology, synteny, and expected rates of synonymous change in coding sequence. Availability and implementation The Pandagma workflows, example configurations, implementation details, and scripts for retrieving public datasets, are available at https://github.com/legumeinfo/pandagma [ABSTRACT FROM AUTHOR]

Histone oxidation provides a direct link between metabolically generated reactive oxygen species (ROS) and chromatin architectural changes enabling the activation of stress response gene expression. Histone oxidation is enacted directly by reactive metabolites and does not depend on enzyme catalysis or cofactor/substrate availability. Residues most susceptible to oxidative modification (cysteine, methionine, and tyrosine) are distinct from those modified by more traditional epigenetic writers (lysine and arginine), providing a distinct and independent pathway for metabolism-driven epigenetic regulation. The emergence of aerobic respiration created unprecedented bioenergetic advantages, while imposing the need to protect critical genetic information from reactive byproducts of oxidative metabolism (i.e., reactive oxygen species, ROS). The evolution of histone proteins fulfilled the need to shield DNA from these potentially damaging toxins, while providing the means to compact and structure massive eukaryotic genomes. To date, several metabolism-linked histone post-translational modifications (PTMs) have been shown to regulate chromatin structure and gene expression. However, whether and how PTMs enacted by metabolically produced ROS regulate adaptive chromatin remodeling remain relatively unexplored. Here, we review novel mechanistic insights into the interactions of ROS with histones and their consequences for the control of gene expression regulation, cellular plasticity, and behavior. [ABSTRACT FROM AUTHOR]

One of the major functions of programmed cell death (apoptosis) is the removal of cells that suffered oncogenic mutations, thereby preventing cancerous transformation. By making use of a Double-Headed-EP (DEP) transposon, a P element derivative made in our laboratory, we made an insertional mutagenesis screen in Drosophila melanogaster to identify genes that, when overexpressed, suppress the p53 -activated apoptosis. The DEP element has Gal4-activatable, outward-directed UAS promoters at both ends, which can be deleted separately in vivo. In the DEP insertion mutants, we used the GMR-Gal4 driver to induce transcription from both UAS promoters and tested the suppression effect on the apoptotic rough eye phenotype generated by an activated UAS- p53 transgene. By DEP insertions, 7 genes were identified, which suppressed the p53 -induced apoptosis. In 4 mutants, the suppression effect resulted from single genes activated by 1 UAS promoter (Pka-R2 , Rga , crol , and Spt5). In the other 3 (Orct2 , Polr2M , and stg), deleting either UAS promoter eliminated the suppression effect. In qPCR experiments, we found that the genes in the vicinity of the DEP insertion also showed an elevated expression level. This suggested an additive effect of the nearby genes on suppressing apoptosis. In the eukaryotic genomes, there are coexpressed gene clusters. Three of the DEP insertion mutants are included, and 2 are in close vicinity of separate coexpressed gene clusters. This raises the possibility that the activity of some of the genes in these clusters may help the suppression of the apoptotic cell death. [ABSTRACT FROM AUTHOR]

Retrotransposons are invasive genetic elements, which replicate by copying and pasting themselves throughout the genome in a process called retrotransposition. The most abundant retrotransposons by number in the human genome are Alu and LINE-1 elements, which comprise approximately 40% of the human genome. The ability of retrotransposons to expand and colonize eukaryotic genomes has rendered them evolutionarily successful and is responsible for creating genetic alterations leading to significant impacts on their hosts. Previous research suggested that hypomethylation of Alu and LINE-1 elements is associated with global hypomethylation and genomic instability in several types of cancer and diseases, such as neurodegenerative diseases, obesity, osteoporosis, and diabetes mellitus (DM). With the advancement of sequencing technologies and computational tools, the study of the retrotransposon's association with physiology and diseases is becoming a hot topic among researchers. Quantifying Alu and LINE-1 methylation is thought to serve as a surrogate measurement of global DNA methylation level. Although Alu and LINE-1 hypomethylation appears to serve as a cellular senescence biomarker promoting genomic instability, there is sparse information available regarding their potential functional and biological significance in DM. This review article summarizes the current knowledge on the involvement of the main epigenetic alterations in the methylation status of Alu and LINE-1 retrotransposons and their potential role as epigenetic markers of global DNA methylation in the pathogenesis of DM. [ABSTRACT FROM AUTHOR]

The WD40 domain is one of the most abundant domains and is among the top interacting domains in eukaryotic genomes. The WD40 domain of ATG16L1 is essential for LC3 recruitment to endolysosomal membranes during non-canonical autophagy, but dispensable for canonical autophagy. Canonical autophagy was utilized by FMDV, while the relationship between FMDV and non-canonical autophagy is still elusive. In the present study, WD40 knockout (KO) PK15 cells were successfully generated via CRISPR/cas9 technology as a tool for studying the effect of non-canonical autophagy on FMDV replication. The results of growth curve analysis, morphological observation and karyotype analysis showed that the WD40 knockout cell line was stable in terms of growth and morphological characteristics. After infection with FMDV, the expression of viral protein, viral titers, and the number of copies of viral RNA in the WD40-KO cells were significantly greater than those in the wild-type PK15 cells. Moreover, RNA‒seq technology was used to sequence WD40-KO cells and wild-type cells infected or uninfected with FMDV. Differentially expressed factors such as Mx1, RSAD2, IFIT1, IRF9, IFITM3, GBP1, CXCL8, CCL5, TNFRSF17 were significantly enriched in the autophagy, NOD-like receptor signaling pathway, RIG-I-like receptor signaling pathway, Toll-like receptor signaling pathway, cytokine-cytokine receptor interaction and TNF signaling pathway, etc. The expression levels of differentially expressed genes were detected via qRT‒PCR, which was consistent with the RNA‒seq data. Here, we experimentally demonstrate for the first time that knockout of the WD40 domain of ATG16L1 enhances FMDV replication by downregulation innate immune factors. In addition, this result also indicates non-canonical autophagy inhibits FMDV replication. In total, our results play an essential role in regulating the replication level of FMDV and providing new insights into virus–host interactions and potential antiviral strategies. Importance: FMDV is a pathogen that causes highly contagious animal disease worldwide. The WD40 domain of ATG16L1 is necessary for non-canonical autophagy, while dispensable for canonical autophagy. Thus, the WD40 knockout PK15 cell line established by present study could use as a tool to research the impact of non-canonical autophagy on FMDV replication. The results showed that knockout of the WD40 domain of ATG16L1 in PK15 cells could enhance FMDV replication. The results also showed that differentially expressed innate immune factors, such as Mx1, IFIT1, RSAD2, and IRF9, were significantly enriched in the interferon signaling pathway. Our research also indicates non-canonical autophagy inhibits FMDV replication by innate immune, which helps to elucidate the role of the non-canonical autophagy in the host antiviral strategies. [ABSTRACT FROM AUTHOR]

Background: As genomes of many eukaryotic species, especially plants, are large and complex, their de novo sequencing and assembly is still a difficult task despite progress in sequencing technologies. An alternative to genome assembly is the assembly of transcriptome, the set of RNA products of the expressed genes. While a bunch of de novo transcriptome assemblers exists, the challenges of transcriptomes (the existence of isoforms, the uneven expression levels across genes) complicates the generation of high-quality assemblies suitable for downstream analyses. Results: We developed Trans2express – a web-based tool and a pipeline of de novo hybrid transcriptome assembly and postprocessing based on rnaSPAdes with a set of subsequent filtrations. The pipeline was tested on Arabidopsis thaliana cDNA sequencing data obtained using Illumina and Oxford Nanopore Technologies platforms and three non-model plant species. The comparison of structural characteristics of the transcriptome assembly with reference Arabidopsis genome revealed the high quality of assembled transcriptome with 86.1% of Arabidopsis expressed genes assembled as a single contig. We tested the applicability of the transcriptome assembly for gene expression analysis. For both Arabidopsis and non-model species the results showed high congruence of gene expression levels and sets of differentially expressed genes between analyses based on genome and based on the transcriptome assembly. Conclusions: We present Trans2express – a protocol for de novo hybrid transcriptome assembly aimed at recovering of a single transcript per gene. We expect this protocol to promote the characterization of transcriptomes and gene expression analysis in non-model plants and web-based tool to be of use to a wide range of plant biologists. [ABSTRACT FROM AUTHOR]

Satellite DNA repeats are repetitive DNA sequences found in eukaryotic genomes, typically consisting of short DNA motifs repeated in tandem arrays. Despite the vast body of literature on satellite DNA repeats in other taxa, investigations specifically targeting Tettigoniidae remain conspicuously absent. Our study aims to fill a critical gap in our understanding of satellitome evolutionary processes shaping Tettigoniidae genomes. Repeatome analysis revealed that the Meconema thalassinum genome comprises 92%, and Phryganogryllacris superangulata had the lowest value of 34%, with an average of 67% in other Tettigoniidae species. The analysis reveals significant variation in the number of satellite DNA repeats across species of the Tettigoniidae family, with M. thalassinum exhibiting the highest count, 246, reported in insects to date and the lowest count, 10, in Pholidoptera griseoptera. Ruspolia dubia and Ruspolia yunnana, which are congeneric species, showcase distinct counts of 104 and 84 families, respectively. Satellite DNA repeats in R. dubia exhibit the highest abundance, constituting 17.2% of the total genome, while the lowest abundance was reported in P. griseoptera, at 5.65%. The genome size correlates weakly with the satellite DNA family count (rs = 0.42, p = 0.29), but a strong correlation exists between satellite abundance and family number (rs = 0.73, p = 0.03). Moreover, the analysis of satellite DNA gain and loss patterns provides insights into the amplification and homogenization of satellite DNA families within the genome, with species-specific repeats exhibiting a positive trend toward amplification. The chromosomal distribution in M. thalassinum displayed that the highest accumulation was observed on Chr12, Chr01, and Chr04, constituting 17.79%, 17.4%, and 17.22% of the total chromosome size, respectively. The chromosome-specific propagation of satellite DNA families was evident, with MthSat01 solely on chromosome 1 and MthSat170 on chromosome 2, sharing 1.64% and 2.33%. The observed conservation and variations in satellite DNA number and abundances, along with distinct patterns of gain and loss, indicate the influence of potentially diverse evolutionary processes shaping the genomic landscape of these insects, which requires further investigation. Furthermore, the differential accumulation of satellite DNA on specific chromosomes implies that potential chromosome-specific functions or structural features influence the retention and proliferation of satellite sequences. [ABSTRACT FROM AUTHOR]

Endogenous viral elements (EVEs) have been reported to exist widely in the genomes of eukaryotic organisms, and they are closely associated with the growth, development, genetics, adaptation, and evolution of their hosts. In this study, two methods—homologous sequence search and genome alignment—were used to explore the endogenous viral sequences in the genomes of Fragaria species. Results revealed abundant endogenous pararetroviruses (EPRVs) in the genomes of Fragaria species, including 786 sequences belonging to five known taxa such as Caulimovirus and other unclassified taxa. Differences were observed in the detected EPRVs between the two methods, with the homologous sequence search having a greater number of EPRVs. On the contrary, genome alignment identified various types and sources of virus-like sequences. Furthermore, through genome alignment, a 267-bp sequence with 95% similarity to the gene encoding the aphid-transmitted protein of Strawberry vein banding virus (Caulimovirus venafragariae) was discovered in the F. chiloensis genome, which was likely a recent insertion. In addition, the statistical analysis of the genome alignment results indicated a remarkably higher abundance of virus-like sequences in the genomes of polyploid strawberries compared with diploid ones. Moreover, the differences in virus-like sequences were observed between the genomes of Fragaria species and those of their close relatives. This study enriched the diversity of viruses that infect strawberries, and laid a theoretical foundation for further research on the origin of endogenous viruses in the strawberry genome, host–virus interactions, adaptation, evolution, and their functions. [ABSTRACT FROM AUTHOR]

Background: Transposable elements (TEs) are major components of eukaryotic genomes. The extensive body of evidence suggests that although they were once considered "genomic parasites", transposons and their transcripts perform specific functions, such as regulation of early embryo development. Understanding the role of TEs in such parasites as trematodes is becoming critically important. Fasciola hepatica, a parasite affecting humans and livestock, undergoes a complex life cycle in diverse environments and hosts, and knowledge about its life cycle regulation is scarce so far. Methods: We summarized the data regarding the repetitive elements in F. hepatica and conducted bulk RNA-seq analysis across its life cycle stages. TE expression profiles were analyzed, focusing on differential expression and potential homology with previously described long non-coding RNAs (lncRNAs). Results: Differential expression analysis revealed stage-specific TE transcription patterns, notably peaking during egg and metacercariae stages. Some TEs showed homology with known lncRNAs and contained putative transcription factor binding sites. Interestingly, TE transcription levels were highest in eggs and metacercariae compared to adults, suggesting regulatory roles in trematode life cycle transitions. Conclusions: These findings suggest that TEs may play roles in regulating trematode life cycle transitions. Moreover, TE homology with lncRNAs underscores their significance in gene regulation. [ABSTRACT FROM AUTHOR]

The CRISPR/Cas technology of targeted genome editing made it possible to carry out genetic engineering manipulations with eukaryotic genomes with a high efficiency. Targeted induction of site-specific DNA breaks is one of the key stages of the technology. The cell repairs the breaks via one of the two pathways, nonhomologous end joining (NHEJ) and homology-driven repair (HDR). The choice of the DNA repair pathway is determined by the architecture of the DNA break region formed as a result of terminal resection and depends on the cell cycle phase. NHEJ is the main pathway of double-strand break (DSB) repair in mammalian cells and involves a nonspecific ligation reaction. The reaction accuracy depends on the structure of break ends, and various insertions or deletions may arise as a result in the target genome region. Integration of a necessary sequence into the genome occurs via HDR, which requires a template with homology regions flanking a DSB. Introducing a genetic construct into a particular genomic locus is an important task, but is currently intricate and laborious to perform. However, the choice of the repair pathway can be of principal importance for basic research of gene functions and construction of animal models of human diseases to develop therapies. The review summarizes and systematizes the available information on strategies designed to increase the HDR efficiency. The strategies that most efficiently shift the balance towards HDR include use of NHEJ inhibitors, regulation of the key factors of homologous recombination, control of the cell cycle and chromatin status, and construction of HDR templates. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes are organized into chromatin domains through long-range chromatin interactions which are mediated by the binding of architectural proteins, such as CTCF and cohesin, and histone modifications. Based on the published Hi-C and ChIP-seq datasets in human monocyte-derived macrophages, we identified 206 and 127 differential chromatin interactions (DCIs) that were not located within transcription readthrough regions in influenza A virus- and interferon β-treated cells, respectively, and found that the binding positions of CTCF and RAD21 within more than half of the DCI sites did not change. However, five histone modifications, H3K4me3, H3K27ac, H3K36me3, H3K9me3, and H3K27me3, showed significantly more dramatic changes than CTCF and RAD21 within the DCI sites. For H3K4me3, H3K27ac, H3K36me3, and H3K27me3, significantly more dramatic changes were observed outside than within the DCI sites. We further applied a motif scanning approach to discover proteins that might correlate with changes in histone modifications and chromatin interactions and found that PRDM9, ZNF384, and STAT2 frequently bound to DNA sequences corresponding to 1 kb genomic intervals with gains or losses of a histone modification within the DCI sites. This study explores the dynamic regulation of chromatin interactions and extends the current knowledge of the relationship between histone modifications and chromatin interactions. [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]

Simple Summary: Most MAGE family genes have been identified on the mammalian genome and are located on the X chromosome. These genes are classified into type I and type II based on their sequence and function. DreNDN was the root of this evolutionary tree. In pigs, type I MAGE genes are predominantly expressed in the testis tissue, and type II MAGE genes are primarily expressed in the brain tissue. These findings are a valuable resource for acquiring insight into the evolution and expression of the MAGE family genes. The melanoma-associated antigen (MAGE) family found in eukaryotes plays a crucial role in cell proliferation and differentiation, spermatogenesis, neural development, etc. This study explored the validation and evolution of MAGE genes in eukaryotic genomes and their distribution and expression patterns in pigs. In total, 249 MAGE genes were found on 13 eukaryotic species. In total, 33, 25, and 18 genes were located on human, mouse, and pig genomes, respectively. We found eight, four, and three tandemly duplicated gene clusters on the human, mouse, and pig genomes, respectively. The majority of MAGE genes in mammals are located on the X chromosome. According to the phylogenetic analysis, the MAGE family genes were classified into 11 subfamilies. The NDN gene in zebrafish (DreNDN) was the root of this evolutionary tree. In total, 10 and 11 MAGE genes on human and mouse genomes, respectively, exhibited a collinearity relationship with the MAGE genes on pig genomes. Taking the MAGE family genes in pigs, the MAGE subfamilies had similar gene structures, protein motifs, and biochemical attributes. Using the RNA-seq data of Duroc pigs and Rongchang pigs, we detected that the expression of type I MAGE genes was higher in reproductive tissues, but type II MAGE genes were predominantly expressed in the brain tissue. These findings are a valuable resource for gaining insight into the evolution and expression of the MAGE family genes. [ABSTRACT FROM AUTHOR]

This article discusses the concept of pangenomics and its relevance to the study of human fungal pathogens. Pangenomes are the total collection of genes within a given phylogenetic group, consisting of a core genome and an accessory genome. The accessory genome is hypothesized to be involved in fungal adaptation to the host or environment, as well as in communication, pathogenicity, and antifungal resistance. Pangenomes can help analyze genomic differences between strains of the same species and provide a reference for experimental studies. They also reduce bias introduced by single reference genomes and elucidate phenotypic variation within a species. The article highlights the importance of functional annotation and experimental characterization of accessory genes, as well as the challenges of genomic data availability and quality, and the lack of a methodological "gold standard" and data curation. Overall, fungal pangenomes have the potential to reveal novel insights into the evolution, pathogenesis, and phenotypic heterogeneity of human fungal pathogens. [Extracted from the article]

5-Methylcytosine (5mC) is a widespread silencing mechanism that controls genomic parasites. In eukaryotes, 5mC has gained complex roles in gene regulation beyond parasite control, yet 5mC has also been lost in many lineages. The causes for 5mC retention and its genomic consequences are still poorly understood. Here, we show that the protist closely related to animals Amoebidium appalachense features both transposon and gene body methylation, a pattern reminiscent of invertebrates and plants. Unexpectedly, hypermethylated genomic regions in Amoebidium derive from viral insertions, including hundreds of endogenized giant viruses, contributing 14% of the proteome. Using a combination of inhibitors and genomic assays, we demonstrate that 5mC silences these giant virus insertions. Moreover, alternative Amoebidium isolates show polymorphic giant virus insertions, highlighting a dynamic process of infection, endogenization, and purging. Our results indicate that 5mC is critical for the controlled coexistence of newly acquired viral DNA into eukaryotic genomes, making Amoebidium a unique model to understand the hybrid origins of eukaryotic DNA. [ABSTRACT FROM AUTHOR]

Background: Structural variations play an important role in bacterial genomes. They can mediate genome adaptation quickly in response to the external environment and thus can also play a role in antibiotic resistance. The detection of structural variations in bacteria is challenging, and the recognition of even small rearrangements can be important. Even though most detection tools are aimed at and benchmarked on eukaryotic genomes, they can also be used on prokaryotic genomes. The key features of detection are the ability to detect small rearrangements and support haploid genomes. Because of the limiting performance of a single detection tool, combining the detection abilities of multiple tools can lead to more robust results. There are already available workflows for structural variation detection for long-reads technologies and for the detection of single-nucleotide variation and indels, both aimed at bacteria. Yet we are unaware of structural variations detection workflows for the short-reads sequencing platform. Motivated by this gap we created our workflow. Further, we were interested in increasing the detection performance and providing more robust results. Results: We developed an open-source bioinformatics pipeline, ProcaryaSV, for the detection of structural variations in bacterial isolates from paired-end short sequencing reads. Multiple tools, starting with quality control and trimming of sequencing data, alignment to the reference genome, and multiple structural variation detection tools, are integrated. All the partial results are then processed and merged with an in-house merging algorithm. Compared with a single detection approach, ProcaryaSV has improved detection performance and is a reproducible easy-to-use tool. Conclusions: The ProcaryaSV pipeline provides an integrative approach to structural variation detection from paired-end next-generation sequencing of bacterial samples. It can be easily installed and used on Linux machines. It is publicly available on GitHub at https://github.com/robinjugas/ProcaryaSV. [ABSTRACT FROM AUTHOR]

Motivation The gene content regulates the biology of an organism. It varies between species and between individuals of the same species. Although tools have been developed to identify gene content changes in bacterial genomes, none is applicable to collections of large eukaryotic genomes such as the human pangenome. Results We developed pangene, a computational tool to identify gene orientation, gene order, and gene copy-number changes in a collection of genomes. Pangene aligns a set of input protein sequences to the genomes, resolves redundancies between protein sequences and constructs a gene graph with each genome represented as a walk in the graph. It additionally finds subgraphs, which we call bibubbles, that capture gene content changes. Applied to the human pangenome, pangene identifies known gene-level variations and reveals complex haplotypes that are not well studied before. Pangene also works with high-quality bacterial pangenome and reports similar numbers of core and accessory genes in comparison to existing tools. Availability and implementation Source code at https://github.com/lh3/pangene ; prebuilt pangene graphs can be downloaded from https://zenodo.org/records/8118576 and visualized at https://pangene.bioinweb.org [ABSTRACT FROM AUTHOR]

Motivation Gene clusters, defined as a set of genes encoding functionally related proteins, are abundant in eukaryotic genomes. Despite the increasing availability of chromosome-level genomes, the comprehensive analysis of gene family evolution remains largely unexplored, particularly for large and highly dynamic gene families or those including very recent family members. These challenges stem from limitations in genome assembly contiguity, particularly in repetitive regions such as large gene clusters. Recent advancements in sequencing technology, such as long reads and chromatin contact mapping, hold promise in addressing these challenges. Results To facilitate the identification, analysis, and visualization of physically clustered gene family members within chromosome-level genomes, we introduce GALEON, a user-friendly bioinformatic tool. GALEON identifies gene clusters by studying the spatial distribution of pairwise physical distances among gene family members along with the genome-wide gene density. The pipeline also enables the simultaneous analysis and comparison of two gene families and allows the exploration of the relationship between physical and evolutionary distances. This tool offers a novel approach for studying the origin and evolution of gene families. Availability and implementation GALEON is freely available from https://www.ub.edu/softevol/galeon and https://github.com/molevol-ub/galeon [ABSTRACT FROM AUTHOR]

Eukaryotic genomes store information on many levels, including their linear DNA sequence, the posttranslational modifications of its constituents (epigenetic modifications), and its three-dimensional folding. Understanding how this information is stored and read requires multidisciplinary collaborations from many branches of science beyond biology, including physics, chemistry, and computer science. Concurrent recent developments in all these areas have enabled researchers to image the genome with unprecedented spatial and temporal resolution. In this review, we focus on what single-molecule imaging and tracking of individual proteins in live cells have taught us about chromatin structure and dynamics. Starting with the basics of single-molecule tracking (SMT), we describe some advantages over in situ imaging techniques and its current limitations. Next, we focus on single-nucleosome studies and what they have added to our current understanding of the relationship between chromatin dynamics and transcription. In celebration of Robert Feulgen's ground-breaking discovery that allowed us to start seeing the genome, we discuss current models of chromatin structure and future challenges ahead. [ABSTRACT FROM AUTHOR]

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]

Copyright of Advances in Biochemistry / Postepy Biochemii is the property of Polish Biochemical Society / Acta Biochimica Polonica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Understanding the function of eukaryotic genomes, including the human genome, is undoubtedly one of the major scientific challenges of the 21st century. The cornerstone of eukaryotic genome organization is nucleosomes—elementary building blocks of chromatin about 10 nm in size that wrap DNA around an octamer of histone proteins. Nucleosomes are integral players in all genomic processes, including transcription, DNA replication and repair. They mediate genome regulation at the epigenetic level, bridging the discrete nature of the genetic information encoded in DNA with the analog physical nature of the intermolecular interactions required to access that information. Due to their relatively large size and dynamic nature, nucleosomes are difficult objects for experimental characterization. Molecular dynamics (MD) simulations have emerged over the years as a useful tool to complement experimental studies. Particularly in recent years, advances in computing power, refinement of MD force fields and codes have opened up new frontiers in terms of simulation timescales and quality for nucleosomes and related systems. It has become possible to elucidate in atomistic detail their functional dynamics modes such as DNA unwrapping and sliding, to characterize the effects of epigenetic modifications, DNA and protein sequence variation on nucleosome structure and stability, to describe the mechanisms governing nucleosome interactions with chromatin‐associated proteins and the formation of supranucleosome structures. In this review, we systematically analyzed all‐atom MD simulation studies of nucleosomes and related structures published since 2018 and discussed their relevance in the context of older studies, experimental data, and related coarse‐grained and multiscale studies. This article is categorized under:Software > Molecular ModelingMolecular and Statistical Mechanics > Molecular Dynamics and Monte‐Carlo MethodsStructure and Mechanism > Computational Biochemistry and Biophysics [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Transposon-derived introns as an element shaping the structure of eukaryotic genomes" published in the journal Mobile DNA. The correction addresses an error in Figure 2 and its legend, clarifying that the intron insertion shown is a hypothetical mechanism that has not been experimentally proven. The corrected figure provides a hypothesized mechanism of intron gain assisted by target site duplication. The authors state that the actual mechanisms of introner insertion are not known. [Extracted from the article]

Despite the fact that introns mean an energy and time burden for eukaryotic cells, they play an irreplaceable role in the diversification and regulation of protein production. As a common feature of eukaryotic genomes, it has been reported that in protein-coding genes, the longest intron is usually one of the first introns. The goal of our work was to find a possible difference in the biological function of genes that fulfill this common feature compared to genes that do not. Data on the lengths of all introns in genes were extracted from the genomes of six vertebrates (human, mouse, koala, chicken, zebrafish and fugu) and two other model organisms (nematode worm and arabidopsis). We showed that more than 40% of protein-coding genes have the relative position of the longest intron located in the second or third tertile of all introns. Genes divided according to the relative position of the longest intron were found to be significantly increased in different KEGG pathways. Genes with the longest intron in the first tertile predominate in a range of pathways for amino acid and lipid metabolism, various signaling, cell junctions or ABC transporters. Genes with the longest intron in the second or third tertile show increased representation in pathways associated with the formation and function of the spliceosome and ribosomes. In the two groups of genes defined in this way, we further demonstrated the difference in the length of the longest introns and the distribution of their absolute positions. We also pointed out other characteristics, namely the positive correlation between the length of the longest intron and the sum of the lengths of all other introns in the gene and the preservation of the exact same absolute and relative position of the longest intron between orthologous genes. [ABSTRACT FROM AUTHOR]

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we develop CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5' capped, full-length transcripts. In our study, we evaluate the performance of CapTrap-seq alongside other widely used RNA-seq library preparation protocols in human and mouse tissues, employing both ONT and PacBio sequencing technologies. To explore the quantitative capabilities of CapTrap-seq and its accuracy in reconstructing full-length RNA molecules, we implement a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5'cap formation. Our benchmarks, incorporating the Long-read RNA-seq Genome Annotation Assessment Project (LRGASP) data, demonstrate that CapTrap-seq is a competitive, platform-agnostic RNA library preparation method for generating full-length transcript sequences. Long-read RNA sequencing has the potential to support exhaustive annotation of eukaryotic genomes, but reliable end-to-end sequencing is still challenging. Here the authors report a method called CapTrap-seq which combines the cap-trapping strategy with oligo(dT) priming for precise full-length transcript detection. [ABSTRACT FROM AUTHOR]

Homologous recombination is a key process that governs the stability of eukaryotic genomes during DNA replication and repair. Multiple auxiliary factors regulate the choice of homologous recombination pathway in response to different types of replication stress. Using Schizosaccharomyces pombe we have previously suggested the role of DNA translocases Rrp1 and Rrp2, together with Srs2 helicase, in the common synthesis-dependent strand annealing sub-pathway of homologous recombination. Here we show that all three proteins are important for completion of replication after hydroxyurea exposure and provide data comparing the effect of overproduction of Srs2 with Rrp1 and Rrp2. We demonstrate that Srs2 localises to rDNA region and is required for proper replication of rDNA arrays. Upregulation of Srs2 protein levels leads to enhanced replication stress, chromosome instability and viability loss, as previously reported for Rrp1 and Rrp2. Interestingly, our data suggests that dysregulation of Srs2, Rrp1 and Rrp2 protein levels differentially affects checkpoint response: overproduction of Srs2 activates simultaneously DNA damage and replication stress response checkpoints, while cells overproducing Rrp1 mainly launch DNA damage checkpoint. On the other hand, upregulation of Rrp2 primarily leads to replication stress response checkpoint activation. Overall, we propose that Srs2, Rrp1 and Rrp2 have important and at least partially independent functions in the maintenance of distinct difficult to replicate regions of the genome. [ABSTRACT FROM AUTHOR]

Introduction: Eukaryotic genomes are composed of simple, repetitive sequences, including satellite DNAs (satDNA), which are noncoding sequences arranged in tandem arrays. These sequences play a crucial role in genomic functions and innovations, influencing processes such as the maintenance of nuclear material, the formation of heterochromatin and the differentiation of sex chromosomes. In this genomic era, advances in next-generation sequencing and bioinformatics tools have facilitated the exhaustive cataloging of repetitive elements in genomes, particularly in non-model species. This study focuses on the satDNA content of Ancistrus sp., a diverse species of fish from the Loricariidae family. The genus Ancistrus shows significant karyotypic evolution, with extensive variability from the ancestral diploid number. Methods: By means of bioinformatic approaches, 40 satDNA families in Ancistrus sp., constituting 5.19% of the genome were identified. Analysis of the abundance and divergence landscape revealed diverse profiles, indicating recent amplification and homogenization of these satDNA sequences. Results: The most abundant satellite, AnSat1-142, constitutes 2.1% of the genome, while the least abundant, AnSat40-52, represents 0.0034%. The length of the monomer repeat varies from 16 to 142 base pairs, with an average length of 61 bp. These results contribute to understanding the genomic dynamics and evolution of satDNAs in Ancistrus sp. Conclusion: The study underscores the variability of satDNAs between fish species and provides valuable information on chromosome organization and the evolution of repetitive elements in non-model organisms. [ABSTRACT FROM AUTHOR]

Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations. [ABSTRACT FROM AUTHOR]

Exceptions to Mendelian inheritance often highlight novel chromosomal behaviors. The maize Pl1-Rhoades allele conferring plant pigmentation can display inheritance patterns deviating from Mendelian expectations in a behavior known as paramutation. However, the chromosome features mediating such exceptions remain unknown. Here we show that small RNA production reflecting RNA polymerase IV function within a distal downstream set of five tandem repeats is coincident with meiotically-heritable repression of the Pl1-Rhoades transcription unit. A related pl1 haplotype with three, but not one with two, repeat units also displays the trans-homolog silencing typifying paramutations. 4C interactions, CHD3a-dependent small RNA profiles, nuclease sensitivity, and polyadenylated RNA levels highlight a repeat subregion having regulatory potential. Our comparative and mutant analyses show that transcriptional repression of Pl1-Rhoades correlates with 24-nucleotide RNA production and cytosine methylation at this subregion indicating the action of a specific DNA-dependent RNA polymerase complex. These findings support a working model in which pl1 paramutation depends on trans-chromosomal RNA-directed DNA methylation operating at a discrete cis-linked and copy-number-dependent transcriptional regulatory element. Author summary: Although switching genes "on" and "off" in a coordinated fashion is essential to organismal development and homeostasis, some genes ‐ or specific forms thereof ‐ display unusual switching behaviors where the "off" state is sexually transmitted to offspring. Further, only "off" states persist when both "on" and "off" states are combined in heterozygous condition. Examples of this unusual behavior, known as paramutation, exist for certain transgenes and/or endogenous genes in both plants and animals but the chromosome features triggering this switching and the responsible cellular machinery are poorly understood. Here, we used long-read DNA sequencing, comparative genomics, DNA -DNA interaction assays, mutant analyses, and cytosine methylation profiling to identify a switchable regulatory sequence associated with paramutation at the maize purple plant1 (pl1) locus. These key sequences are embedded within a 5-copy tandem repeat array of mostly transposable elements ~14 kilobases distal to the pl1 gene. The "off" state is associated with these sequences producing so-called "small interfering RNAs" (siRNAs) that may direct the recruitment of chromatin-modifying enzymes. Our findings support a molecular model for paramutation and highlight the regulatory importance of repeated sequences found in large eukaryotic genomes. [ABSTRACT FROM AUTHOR]

Ribosomal DNA (rDNA) contains highly conserved, specifically organized sequences encoding ribosomal RNAs (rRNAs) separated by variable nontranscribed intergenic spacers (NTSs) and is abundant in eukaryotic genomes. These characteristics make the rDNA an informative molecular target to study genome organization, molecular evolution, and phylogenetics. In this study, we characterized the 5S rDNA repeats in the greater duckweed Spiroldela polyrhiza, a species known for its small size, rapid growth, highly conserved genome organization, and low mutation rate. Sequence analysis of at least 12 individually cloned PCR fragments containing the 5S rDNA units for each of six ecotypes that originated from Europe (Ukraine) and Asia (China) revealed two distinct types of 5S rDNA repeats containing NTSs of different lengths and nucleotide compositions. The shorter 5S rDNA repeat units had a highly homogeneous 400-bp NTS, with few ecotype-or region-specific singlenucleotide polymorphisms (SNPs). The longer 5S rDNA units had NTSs of 1056-1084 bp with characteristic intra- and inter-genomic variants due to specific SNPs and insertions/deletions of 4-15-bp DNA elements. We also detected significant variability in the ratio of short/long 5S rDNA variants between ecotypes of S. polyrhiza. The contrasting dynamics of the two types of 5S rDNA units, combined with the unusually low repeat copy number (for plants) in S. polyrhiza (46-220 copies per genome), shows that this species could serve as an excellent model for examining the mechanisms of concerted evolution and functional significance of rDNA variability. [ABSTRACT FROM AUTHOR]

Motivation Identifying chromatin accessibility is one of the key steps in studying the regulation of eukaryotic genomes. The combination of exogenous methyltransferase and nanopore sequencing provides an strategy to identify open chromatin over long genomic ranges at the single-molecule scale. However, endogenous methylation, non-open-chromatin-specific exogenous methylation and base-calling errors limit the accuracy and hinders its application to complex genomes. Results We systematically evaluated the impact of these three influence factors, and developed a model-based computational method, methyltransferase accessible genome region finder (MAGNIFIER), to address the issues. By incorporating control data, MAGNIFIER attenuates the three influence factors with data-adaptive comparison strategy. We demonstrate that MAGNIFIER is not only sensitive to identify the open chromatin with much improved accuracy, but also able to detect the chromatin accessibility of repetitive regions that are missed by NGS-based methods. By incorporating long-read RNA-seq data, we revealed the association between the accessible Alu elements and non-classic gene isoforms. Availability and implementation Freely available on web at https://github.com/Goatofmountain/MAGNIFIER. [ABSTRACT FROM AUTHOR]

Horizontal genetic transfer (HGT) is a common phenomenon in eukaryotic genomes. However, the mechanisms by which HGT‐derived genes persist and integrate into other pathways remain unclear. This topic is of significant interest because, over time, the stressors that initially favoured the fixation of HGT may diminish or disappear. Despite this, the foreign genes may continue to exist if they become part of a broader stress response or other pathways. The conventional model suggests that the acquisition of HGT equates to adaptation. However, this model may evolve into more complex interactions between gene products, a concept we refer to as the 'Integrated HGT Model' (IHM). To explore this concept further, we studied specialized HGT‐derived genes that encode heavy metal detoxification functions. The recruitment of these genes into other pathways could provide clear examples of IHM. In our study, we exposed two anciently diverged species of polyextremophilic red algae from the Galdieria genus to arsenic and mercury stress in laboratory cultures. We then analysed the transcriptome data using differential and coexpression analysis. Our findings revealed that mercury detoxification follows a 'one gene‐one function' model, resulting in an indivisible response. In contrast, the arsH gene in the arsenite response pathway demonstrated a complex pattern of duplication, divergence and potential neofunctionalization, consistent with the IHM. Our research sheds light on the fate and integration of ancient HGTs, providing a novel perspective on the ecology of extremophiles. [ABSTRACT FROM AUTHOR]

Ribonucleotides represent the most common non-canonical nucleotides found in eukaryotic genomes. The sources of chromosome-embedded ribonucleotides and the mechanisms by which unrepaired rNMPs trigger genome instability and human pathologies are not fully understood. The available sequencing technologies only allow to indirectly deduce the genomic location of rNMPs. Oxford Nanopore Technologies (ONT) may overcome such limitation, revealing the sites of rNMPs incorporation in genomic DNA directly from raw sequencing signals. We synthesized two types of DNA molecules containing rNMPs at known or random positions and we developed data analysis pipelines for DNA-embedded ribonucleotides detection by ONT. We report that ONT can identify all four ribonucleotides incorporated in DNA by capturing rNMPs-specific alterations in nucleotide alignment features, current intensity, and dwell time. We propose that ONT may be successfully employed to directly map rNMPs in genomic DNA and we suggest a strategy to build an ad hoc basecaller to analyse native genomes. This study reports the direct detection of ribonucleotides embedded in DNA molecules by Oxford Nanopore sequencing, based on ribonucleotides-induced alterations in basecalling features, currents, and dwell times. [ABSTRACT FROM AUTHOR]

Eukaryotic genomes are compacted and organized into distinct three-dimensional (3D) structures, which range from small-scale nucleosome arrays to large-scale chromatin domains. These chromatin structures play an important role in the regulation of transcription and other nuclear processes. The molecular mechanisms that drive the formation of chromatin structures across scales and the relationship between chromatin structure and function remain incompletely understood. Because the processes involved are complex and interconnected, it is often challenging to dissect the underlying principles in the nuclear environment. Therefore, in vitro reconstitution systems provide a valuable approach to gain insight into the molecular mechanisms by which chromatin structures are formed and to determine the cause-consequence relationships between the processes involved. In this review, we give an overview of in vitro approaches that have been used to study chromatin structures across scales and how they have increased our understanding of the formation and function of these structures. We start by discussing in vitro studies that have given insight into the mechanisms of nucleosome positioning. Next, we discuss recent efforts to reconstitute larger-scale chromatin domains and loops and the resulting insights into the principles of genome organization. We conclude with an outlook on potential future applications of chromatin reconstitution systems and how they may contribute to answering open questions concerning chromatin architecture. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are major components of eukaryotic genomes and are implicated in a range of evolutionary processes. Yet, TE annotation and characterization remain challenging, particularly for nonspecialists, since existing pipelines are typically complicated to install, run, and extract data from. Current methods of automated TE annotation are also subject to issues that reduce overall quality, particularly (i) fragmented and overlapping TE annotations, leading to erroneous estimates of TE count and coverage, and (ii) repeat models represented by short sections of total TE length, with poor capture of 5′ and 3′ ends. To address these issues, we present Earl Grey, a fully automated TE annotation pipeline designed for user-friendly curation and annotation of TEs in eukaryotic genome assemblies. Using nine simulated genomes and an annotation of Drosophila melanogaster , we show that Earl Grey outperforms current widely used TE annotation methodologies in ameliorating the issues mentioned above while scoring highly in benchmarking for TE annotation and classification and being robust across genomic contexts. Earl Grey provides a comprehensive and fully automated TE annotation toolkit that provides researchers with paper-ready summary figures and outputs in standard formats compatible with other bioinformatics tools. Earl Grey has a modular format, with great scope for the inclusion of additional modules focused on further quality control and tailored analyses in future releases. [ABSTRACT FROM AUTHOR]

The special issue of the Journal of Eukaryotic Microbiology focuses on microsporidia, unicellular parasites that infect both vertebrate and invertebrate hosts. With about 220 genera and 1700 species, microsporidia are classified based on ultrastructural features, developmental cycle, and molecular analysis. The issue covers various aspects of microsporidia biology, including genome evolution, gene prediction, taxonomy, host interactions, and pathogenicity. Researchers have made significant contributions to understanding these pathogens, with potential therapeutic targets being explored for diseases like microsporidiosis. [Extracted from the article]

The genomes of cellular organisms display CpG and TpA dinucleotide composition biases. Such biases have been poorly investigated in dsDNA viruses. Here, we show that in dsDNA virus, bacterial, and eukaryotic genomes, the representation of TpA and CpG dinucleotides is strongly dependent on genomic G + C content. Thus, the classical observed/expected ratios do not fully capture dinucleotide biases across genomes. Because a larger portion of the variance in TpA frequency was explained by G + C content, we explored which additional factors drive the distribution of CpG dinucleotides. Using the residuals of the linear regressions as a measure of dinucleotide abundance and ancestral state reconstruction across eukaryotic and prokaryotic virus trees, we identified an important role for phylogeny in driving CpG representation. Nonetheless, phylogenetic ANOVA analyses showed that few host associations also account for significant variations. Among eukaryotic viruses, most significant differences were observed between arthropod‐infecting viruses and viruses that infect vertebrates or unicellular organisms. However, an effect of viral DNA methylation status (either driven by the host or by viral‐encoded methyltransferases) is also likely. Among prokaryotic viruses, cyanobacteria‐infecting phages resulted to be significantly CpG‐depleted, whereas phages that infect bacteria in the genera Burkolderia and Staphylococcus were CpG‐rich. Comparison with bacterial genomes indicated that this effect is largely driven by the general tendency for phages to resemble the host's genomic CpG content. Notably, such tendency is stronger for temperate than for lytic phages. Our data shed light into the processes that shape virus genome composition and inform manipulation strategies for biotechnological applications. [ABSTRACT FROM AUTHOR]