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Transfer RNA (tRNA) contains modified nucleosides essential for modulating protein translation. One of these modifications is queuosine (Q), which affects NAU codons translation rate. For decades, multiple studies have reported a wide variety of species-specific Q-related phenotypes in different eukaryotes, hindering the identification of a general underlying mechanism behind that phenotypic diversity. Here, through bioinformatics analysis of representative eukaryotic genomes we have predicted: i) the genes enriched in NAU codons, whose translation would be affected by tRNA Q-modification (Q-genes); and ii) the specific biological processes of each organism enriched in Q-genes, which generally in eukaryotes would be related to ubiquitination, phosphatidylinositol metabolism, splicing, DNA repair or cell cycle. These bioinformatics results provide evidence to support for the first time in eukaryotes that the wide diversity of phenotypes associated with tRNA Q-modification previously described in various species would directly depend on the control of Q-genes translation, and would allow prediction of unknown Q-dependent processes, such as Akt activation and p53 expression, which we have tested in human cancer cells. Considering the relevance of the Q-related processes, our findings may support further exploration of the role of Q in cancer and other pathologies. Moreover, since eukaryotes must salvage Q from bacteria, we suggest that changes in Q supply by the microbiome would affect the expression of host Q-genes, altering its physiology. [ABSTRACT FROM AUTHOR]

The assembly of repressive heterochromatin in eukaryotic genomes is crucial for silencing lineage-inappropriate genes and repetitive DNA elements. Paradoxically, transcription of repetitive elements within constitutive heterochromatin domains is required for RNA-based mechanisms, such as the RNAi pathway, to target heterochromatin assembly proteins. However, the mechanism by which heterochromatic repeats are transcribed has been unclear. Using fission yeast, we show that the conserved trimeric transcription factor (TF) PhpCNF-Y complex can infiltrate constitutive heterochromatin via its histone-fold domains to transcribe repeat elements. PhpCNF-Y collaborates with a Zn-finger containing TF to bind repeat promoter regions with CCAAT boxes. Mutating either the TFs or the CCAAT binding site disrupts the transcription of heterochromatic repeats. Although repeat elements are transcribed from both strands, PhpCNF-Y-dependent transcripts originate from only one strand. These TF-driven transcripts contain multiple cryptic introns which are required for the generation of small interfering RNAs (siRNAs) via a mechanism involving the spliceosome and RNAi machinery. Our analyses show that siRNA production by this TF-mediated transcription pathway is critical for heterochromatin nucleation at target repeat loci. This study reveals a mechanism by which heterochromatic repeats are transcribed, initiating their own silencing by triggering a primary cascade that produces siRNAs necessary for heterochromatin nucleation. This study shows that the PhpCNF-Y transcription factor infiltrates heterochromatin to produce transcripts with cryptic introns that initiate RNAi-mediated heterochromatin assembly, investigating how heterochromatic repeats initiate their own silencing. [ABSTRACT FROM AUTHOR]

Short tandem repeats (STRs) have emerged as important and hypermutable sites where genetic variation correlates with gene expression in plant and animal systems. Recently, it has been shown that a broad range of transcription factors (TFs) are affected by STRs near or in the DNA target binding site. Despite this, the distribution of STR motif repetitiveness in eukaryote genomes is still largely unknown. Here, we identify monomer and dimer STR motif repetitiveness in 5.1 billion 10-bp windows upstream of translation starts and downstream of translation stops in 25 million genes spanning 1270 species across the eukaryotic Tree of Life. We report that all surveyed genomes have gene-proximal shifts in motif repetitiveness. Within genomes, variation in gene-proximal repetitiveness landscapes correlated to the function of genes; genes with housekeeping functions were depleted in upstream and downstream repetitiveness. Furthermore, the repetitiveness landscapes correlated with TF binding sites, indicating that gene function has evolved in conjunction with cis-regulatory STRs and TFs that recognize repetitive sites. These results suggest that the hypermutability inherent to STRs is canalized along the genome sequence and contributes to regulatory and eco-evolutionary dynamics in all eukaryotes. Despite the abundance of simple repeats in eukaryote genomes, their spatial distribution is largely unknown across taxa. Here, the authors show that repeats accumulate proximally to genes and correlate with gene function and transcription factor profiles, suggesting regulatory roles across eukaryotes. [ABSTRACT FROM AUTHOR]

Background: Long terminal repeats (LTRs) represent important parts of LTR retrotransposons and retroviruses found in high copy numbers in a majority of eukaryotic genomes. LTRs contain regulatory sequences essential for the life cycle of the retrotransposon. Previous experimental and sequence studies have provided only limited information about LTR structure and composition, mostly from model systems. To enhance our understanding of these key sequence modules, we focused on the contrasts between LTRs of various retrotransposon families and other genomic regions. Furthermore, this approach can be utilized for the classification and prediction of LTRs. Results: We used machine learning methods suitable for DNA sequence classification and applied them to a large dataset of plant LTR retrotransposon sequences. We trained three machine learning models using (i) traditional model ensembles (Gradient Boosting), (ii) hybrid convolutional/long and short memory network models, and (iii) a DNA pre-trained transformer-based model using k-mer sequence representation. All three approaches were successful in classifying and isolating LTRs in this data, as well as providing valuable insights into LTR sequence composition. The best classification (expressed as F1 score) achieved for LTR detection was 0.85 using the hybrid network model. The most accurate classification task was superfamily classification (F1=0.89) while the least accurate was family classification (F1=0.74). The trained models were subjected to explainability analysis. Positional analysis identified a mixture of interesting features, many of which had a preferred absolute position within the LTR and/or were biologically relevant, such as a centrally positioned TATA-box regulatory sequence, and TG..CA nucleotide patterns around both LTR edges. Conclusions: Our results show that the models used here recognized biologically relevant motifs, such as core promoter elements in the LTR detection task, and a development and stress-related subclass of transcription factor binding sites in the family classification task. Explainability analysis also highlighted the importance of 5'- and 3'- edges in LTR identity and revealed need to analyze more than just dinucleotides at these ends. Our work shows the applicability of machine learning models to regulatory sequence analysis and classification, and demonstrates the important role of the identified motifs in LTR detection. [ABSTRACT FROM AUTHOR]

The R-loop is a common transcriptional by-product that consists of an RNA-DNA duplex joined to a displaced strand of genomic DNA. While the effects of R-loops on health and disease are well established, there is still an incomplete understanding of the cellular processes responsible for their removal from eukaryotic genomes. Here, we show that a core regulator of chromosome architecture --the Smc5/6 complex-- plays a crucial role in the removal of R-loop structures formed during gene transcription. Consistent with this, budding yeast mutants defective in the Smc5/6 complex and enzymes involved in R-loop resolution show strong synthetic interactions and accumulate high levels of RNA-DNA hybrid structures in their chromosomes. Importantly, we demonstrate that the Smc5/6 complex acts on specific types of RNA-DNA hybrid structures in vivo and promotes R-loop degradation by the RNase H2 enzyme in vitro. Collectively, our results reveal a crucial role for the Smc5/6 complex in the removal of toxic R-loops formed at highly transcribed genes and telomeres. [ABSTRACT FROM AUTHOR]

Introns are highly prevalent in most eukaryotic genomes. Despite the accumulating evidence for benefits conferred by the possession of introns, their specific roles and functions, as well as the processes shaping their evolution, are still only partially understood. Here, we explore the evolution of the eukaryotic intron–exon gene structure by focusing on several key features such as the intron length, the number of introns, and the intron-to-exon length ratio in protein-coding genes. We utilize whole-genome data from 590 species covering the main eukaryotic taxonomic groups and analyze them within a statistical phylogenetic framework. We found that the basic gene structure differs markedly among the main eukaryotic groups, with animals, and particularly chordates, displaying intron-rich genes, compared with plants and fungi. Reconstruction of gene structure evolution suggests that these differences evolved prior to the divergence of the main phyla and have remained mostly conserved within groups. We revisit the previously reported association between the genome size and the mean intron length and report that this association differs considerably among phyla. Analyzing a large and diverse dataset of species with whole-genome information while applying advanced modeling techniques allowed us to obtain a global evolutionary perspective. Our findings may indicate that introns play different molecular and evolutionary roles in different organisms. [ABSTRACT FROM AUTHOR]

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) compose a substantial proportion of the largest eukaryotic genomes. TE diversity has been hypothesized to be negatively correlated with genome size, yet empirical demonstrations of such a relationship in a phylogenetic context are largely lacking. Furthermore, the most abundant type of TEs in genomes varies across groups, and it is not clear if there are patterns of TE activity consistent with genome size among different taxa with large genome sizes. We use low-coverage sequencing of 16 species of Neotropical salamanders, which vary ∼7-fold in genome size, to estimate TE relative abundance and diversity for each species. We also compare the divergence of copies of each TE superfamily to estimate patterns of TE activity in each species. We find a negative relationship between TE diversity and genome size, which is consistent with the hypothesis that either competition among TEs or reduced selection against ectopic recombination may result in lower diversity in the largest genomes. We also find divergent activity patterns in the largest versus the smallest genomes, suggesting that the history of TE activity may explain differences in genome size. Our results suggest that both TE diversity and relative abundance may be predictable, at least within taxonomic groups. [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]

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]

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]

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]

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]

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]

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]

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]

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 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]

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]

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]

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]

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]

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]

DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. To investigate how variation in DNA methylation is regulated on the Z chromosome we utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals. The relatively large numbers of individuals allowed us to identify additional genomic MHM regions on the Z chromosome that were significantly differentially methylated between the sexes. These regions appear to down-regulate local gene expression in males, but not remove it entirely (unlike the lncRNAs identified in the initial MHM regions). These MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. In addition, trans effect hotspots were also identified that were based on the autosomes but affected the Z, and also that were based on the Z chromosome but that affected autosomal DNA methylation regulation. In addition, quantitative trait loci (QTL) that regulate variation in methylation on the Z chromosome, and those loci that regulate methylation on the autosomes that derive from the Z chromosome were mapped. Trans-effect hotspots were also identified that were based on the autosomes but affected the Z, and also one that was based on the Z chromosome but that affected both autosomal and sex chromosome DNA methylation regulation. We show that both cis and trans loci that originate from the Z chromosome never exhibit an interaction with sex, whereas trans loci originating from the autosomes but affecting the Z chromosome always display such an interaction. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes. Author summary: DNA methylation is a key regulator of eukaryote genomes, and is of particular relevance in the regulation of gene expression on the sex chromosomes, with a key role in dosage compensation in mammalian XY systems. In the case of birds, dosage compensation is largely absent, with it being restricted to two small Male Hyper-Methylated (MHM) regions on the Z chromosome. We utilised a wild x domestic advanced intercross in the chicken, with both hypothalamic methylomes and transcriptomes assayed in 124 individuals, to investigate the role that methylation plays in regulating gene expression on the Z chromosome. Our results highlight how additional MHM regions are actually present on the Z chromosome, and they appear to have smaller-scale effects on gene expression in males. Quantitative variation in methylation is also regulated both from the autosomes to the Z chromosome, and from the Z chromosome to the autosomes. In addition, these MHM regions were further tested and the most balanced genes appear to show decreased expression in males, whilst methylation appeared to be far more correlated with gene expression in the less balanced, as compared to the most balanced genes. [ABSTRACT FROM AUTHOR]

The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly insulated in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus. The spatial organization of eukaryotic genomes is linked to their biological functions. Here, the authors study the 3D genome organization of the phytopathogenic fungus Verticillium dahliae, revealing links to evolutionary features conserved throughout the Verticillium genus. [ABSTRACT FROM AUTHOR]

Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing. [ABSTRACT FROM AUTHOR]

Simple Summary: Long terminal repeat (LTR) retrotransposons and non-LTR retrotransposons are two major repetitive components of eukaryotic genomes. Short intersperse elements (SINEs) are non-autonomous non-LTR retrotransposons with a sequence similar to small RNAs such as tRNA and 5S rRNA. Cassandra is a non-autonomous LTR retrotransposon with a 5S rRNA-like sequence in its LTRs. This observation led me to investigate the presence of non-autonomous LTR retrotransposons containing other small RNA sequences in their LTRs. Here, I discover and describe non-autonomous LTR retrotransposons Helenus and Ajax, with LTR regions of respective tRNA and 5S rRNA origin. Helenus and Ajax are likely mobilized by autonomous LTR retrotransposons. Helenus or Ajax are unlikely to encode a functional RNA. The finding of Helenus and Ajax confirms that non-autonomous LTR retrotransposons can be a source of small RNA-like interspersed sequences in the eukaryotic genomes. An understanding of their biology could contribute to the more precise prediction of small RNA genes and cellular RNA components as well as to the understanding of their evolutionary impacts. Terminal repeat retrotransposons in miniature (TRIMs) are short non-autonomous long terminal repeat (LTR) retrotransposons found from various eukaryotes. Cassandra is a unique TRIM lineage which contains a 5S rRNA-derived sequence in its LTRs. Here, two new groups of TRIMs, designated Helenus and Ajax, are reported based on bioinformatics analysis and the usage of Repbase. Helenus is found from fungi, animals, and plants, and its LTRs contain a tRNA-like sequence. It includes two LTRs and between them, a primer-binding site (PBS) and polypurine tract (PPT) exist. Fungal and plant Helenus generate 5 bp target site duplications (TSDs) upon integration, while animal Helenus generates 4 bp TSDs. Ajax includes a 5S rRNA-derived sequence in its LTR and is found from two nemertean genomes. Ajax generates 5 bp TSDs upon integration. These results suggest that despite their unique promoters, Helenus and Ajax are TRIMs whose transposition is dependent on autonomous LTR retrotransposon. These TRIMs can originate through an insertion of SINE in an LTR of TRIM. The discovery of Helenus and Ajax suggests the presence of TRIMs with a promoter for RNA polymerase III derived from a small RNA gene, which is here collectively termed TRIMp3. [ABSTRACT FROM AUTHOR]

Background: Satellite repeats are one of the most rapidly evolving components in eukaryotic genomes and play vital roles in genome regulation, genome evolution, and speciation. As a consequence, the composition, abundance and chromosome distribution of satellite repeats often exhibit variability across various species, genome, and even individual chromosomes. However, we know little about the satellite repeat evolution in allopolyploid genomes. Results: In this study, we investigated the satellite repeat signature in five okra (Abelmoschus esculentus) accessions using genomic and cytogenetic methods. In each of the five accessions, we identified eight satellite repeats, which exhibited a significant level of intraspecific conservation. Through fluorescence in situ hybridization (FISH) experiments, we observed that the satellite repeats generated multiple signals and exhibited variations in copy number across chromosomes. Intriguingly, we found that five satellite repeats were interspersed with centromeric retrotransposons, signifying their involvement in centromeric satellite repeat identity. We confirmed subgenome-biased amplification patterns of these satellite repeats through existing genome assemblies or dual-color FISH, indicating their distinct dynamic evolution in the allotetraploid okra subgenome. Moreover, we observed the presence of multiple chromosomes harboring the 35 S rDNA loci, alongside another chromosomal pair carrying the 5 S rDNA loci in okra using FISH assay. Remarkably, the intensity of 35 S rDNA hybridization signals varied among chromosomes, with the signals predominantly localized within regions of relatively weak DAPI staining, associated with GC-rich heterochromatin regions. Finally, we observed a similar localization pattern between 35 S rDNA and three satellite repeats with high GC content and confirmed their origin in the intergenic spacer region of the 35 S rDNA. Conclusions: Our findings uncover a unique satellite repeat signature in the allotetraploid okra, contributing to our understanding of the composition, abundance, and chromosomal distribution of satellite repeats in allopolyploid genomes, further enriching our understanding of their evolutionary dynamics in complex allopolyploid genomes. [ABSTRACT FROM AUTHOR]

Transposable elements (TEs) are indispensable components of eukaryotic genomes that play diverse roles in gene regulation, recombination, and environmental adaptation. Their ability to mobilize within the genome leads to gene expression and DNA structure changes. TEs serve as valuable markers for genetic and evolutionary studies and facilitate genetic mapping and phylogenetic analysis. They also provide insight into how organisms adapt to a changing environment by promoting gene rearrangements that lead to new gene combinations. These repetitive sequences significantly impact genome structure, function and evolution. This review takes a comprehensive look at TEs and their applications in biotechnology, particularly in the context of plant biology, where they are now considered “genomic gold” due to their extensive functionalities. The article addresses various aspects of TEs in plant development, including their structure, epigenetic regulation, evolutionary patterns, and their use in gene editing and plant molecular markers. The goal is to systematically understand TEs and shed light on their diverse roles in plant biology. [ABSTRACT FROM AUTHOR]

Simple Summary: Transposable elements (TEs) are mobile genetic elements that play vital role in defining and contributing to the size, shape and structure of both prokaryotic and eukaryotic genomes in nature. The piggyBac (PB), a superfamily of DNA transposons, has been isolated over the years from various organisms including insects, fungi and plants. These piggyBac transposon systems have high efficiency with a wide usage in the study of gene therapies, mutagenesis and transgenesis. Currently, there is limited information available on DNA transposons in small (compact) genomes of animals. Therefore, this study aims to annotate the PB transposons in small genomes of animals, revealing their evolution profiles in both vertebrate and invertebrate genomes. TEs, including DNA transposons, are major contributors of genome expansions, and have played a very significant role in shaping the evolution of animal genomes, due to their capacity to jump from one genomic position to the other. In this study, we investigated the evolution landscapes of PB transposons, including their distribution, diversity, activity and structure organization in 79 species of small (compact) genomes of animals comprising both vertebrate and invertebrates. Overall, 212 PB transposon types were detected from almost half (37) of the total number of the small genome species (79) investigated. The detected PB transposon types, which were unevenly distributed in various genera and phyla, have been classified into seven distinct clades or families with good bootstrap support (>80%). The PB transposon types that were identified have a length ranging from 1.23 kb to 9.51 kb. They encode transposases of approximately ≥500 amino acids in length, and possess terminal inverted repeats (TIRs) ranging from 4 bp to 24 bp. Though some of the transposon types have long TIRs (528 bp), they still maintain the consistent and reliable 4 bp target site duplication (TSD) of TTAA. However, PiggyBac-2_Cvir transposon originating from the Crassostrea virginica species exhibits a unique TSD of TATG. The TIRs of the transposons in all the seven families display high divergence, with a highly conserved 5′ end motif. The core transposase domains (DDD) were better conserved among the seven different families compared to the other protein domains, which were less prevalent in the vertebrate genome. The divergent evolution dynamics analysis also indicated that the majority of the PB transposon types identified in this study are either relatively young or old, with some being active. Additionally, numerous invasions of PB transposons were found in the genomes of both vertebrate and invertebrate animals. The data reveals that the PB superfamily is widely distributed in these species. PB transposons exhibit high diversity and activity in the small genomes of animals, and might play a crucial role in shaping the evolution of these small genomes of animals. [ABSTRACT FROM AUTHOR]

Identification of viruses and further assembly of viral genomes from the next-generation-sequencing data are essential steps in virome studies. This study presented a one-stop tool named VIGA (available at https://github.com/viralInformatics/VIGA) for eukaryotic virus identification and genome assembly from NGS data. It was composed of four modules, namely, identification, taxonomic annotation, assembly and novel virus discovery, which integrated several third-party tools such as BLAST, Trinity, MetaCompass and RagTag. Evaluation on multiple simulated and real virome datasets showed that VIGA assembled more complete virus genomes than its competitors on both the metatranscriptomic and metagenomic data and performed well in assembling virus genomes at the strain level. Finally, VIGA was used to investigate the virome in metatranscriptomic data from the Human Microbiome Project and revealed different composition and positive rate of viromes in diseases of prediabetes, Crohn's disease and ulcerative colitis. Overall, VIGA would help much in identification and characterization of viromes, especially the known viruses, in future studies. [ABSTRACT FROM AUTHOR]

A G-quadruplex (G4) is a typical non-B DNA structure and involved in various DNA-templated events in eukaryotic genomes. PEG and PDS chemicals have been widely applied for promoting the folding of in vivo or in vitro G4s. However, how PEG and PDS preferentially affect a subset of G4 formation genome-wide is still largely unknown. We here conducted a BG4-based IP-seq in vitro under K++PEG or K++PDS conditions in the rice genome. We found that PEG-favored IP-G4s+ have distinct sequence features, distinct genomic distributions and distinct associations with TEGs, non-TEGs and subtypes of TEs compared to PDS-favored ones. Strikingly, PEG-specific IP-G4s+ are associated with euchromatin with less enrichment levels of DNA methylation but with more enriched active histone marks, while PDS-specific IP-G4s+ are associated with heterochromatin with higher enrichment levels of DNA methylation and repressive marks. Moreover, we found that genes with PEG-specific IP-G4s+ are more expressed than those with PDS-specific IP-G4s+, suggesting that PEG/PDS-specific IP-G4s+ alone or coordinating with epigenetic marks are involved in the regulation of the differential expression of related genes, therefore functioning in distinct biological processes. Thus, our study provides new insights into differential impacts of PEG and PDS on G4 formation, thereby advancing our understanding of G4 biology. [ABSTRACT FROM AUTHOR]