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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]

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]

[Display omitted] • Premature termination turns over a large fraction of pol II at promoter proximal pause sites. • Integrator binds paused pol II and terminates transcription. • Premature termination occurs by Ubiquitylation and proteasomal degradation of pol II. • Premature termination likely functions both to regulate gene expression and as a quality control mechanism. Eukaryotic genomes are widely transcribed by RNA polymerase II (pol II) both within genes and in intergenic regions. POL II elongation complexes comprising the polymerase, the DNA template and nascent RNA transcript must be extremely processive in order to transcribe the longest genes which are over 1 megabase long and take many hours to traverse. Dedicated termination mechanisms are required to disrupt these highly stable complexes. Transcription termination occurs not only at the 3′ ends of genes once a full length transcript has been made, but also within genes and in promiscuously transcribed intergenic regions. Termination at these latter positions is termed "premature" because it is not triggered in response to a specific signal that marks the 3' end of a gene, like a polyA site. One purpose of premature termination is to remove polymerases from intergenic regions where they are "not wanted" because they may interfere with transcription of overlapping genes or the progress of replication forks. Premature termination has recently been appreciated to occur at surprisingly high rates within genes where it is speculated to serve regulatory or quality control functions. In this review I summarize current understanding of the different mechanisms of premature termination and its potential functions. [ABSTRACT FROM AUTHOR]

Noncoding satellite DNA repeats are abundant at the pericentromeric heterochromatin of eukaryotic chromosomes. During interphase, sequence-specific DNA-binding proteins cluster these repeats from multiple chromosomes into nuclear foci known as chromocenters. Despite the pivotal role of chromocenters in cellular processes like genome encapsulation and gene repression, the associated proteins remain incompletely characterized. Here, we use 2 satellite DNA-binding proteins, D1 and Prod, as baits to characterize the chromocenter-associated proteome in Drosophila embryos, ovaries, and testes through quantitative mass spectrometry. We identify D1- and Prod-associated proteins, including known heterochromatin proteins as well as proteins previously unlinked to satellite DNA or chromocenters, thereby laying the foundation for a comprehensive understanding of cellular functions enabled by satellite DNA repeats and their associated proteins. Interestingly, we find that multiple components of the transposon-silencing piRNA pathway are associated with D1 and Prod in embryos. Using genetics, transcriptomics, and small RNA profiling, we show that flies lacking D1 during embryogenesis exhibit transposon expression and gonadal atrophy as adults. We further demonstrate that this gonadal atrophy can be rescued by mutating the checkpoint kinase, Chk2, which mediates germ cell arrest in response to transposon mobilization. Thus, we reveal that a satellite DNA-binding protein functions during embryogenesis to silence transposons, in a manner that is heritable across later stages of development. Pericentromeric heterochromatin clusters into nuclear foci known as chromocenters, the proteome of which remains largely unknown. This study uses quantitative mass spectrometry to characterize the chromocenter-associated proteome in multiple tissues, identifying a link between satellite DNA-binding proteins and transposon repression during embryogenesis. [ABSTRACT FROM AUTHOR]

The Yadong trout (Salmo trutta), a species endemic to the Yatung River in Tibet, China, was classified as a second-class protected species in the 20th century. Now, it is considered one of the most important fishery resources in China. In this study, we assembled a near-complete genome of the S. trutta, integrating PacBio HiFi, Hi-C, and ONT sequencing technologies. The genome assembly spans 2.49 Gb, with 96.87% of the sequence anchored onto 40 chromosomes. In this assembly, a total of 12 chromosomes were assembled to a gap-free level, with 8 of them reaching the telomere-to-telomere level. The completeness of this assembly was assessed at 99.50% by BUSCO, containing approximately 63.24% repetitive sequences, and predicted to encode 41,782 protein-coding genes. This is the first near-complete genome assembly of the S. trutta, providing an essential resource for molecular breeding and germplasm conservation of this important species. [ABSTRACT FROM AUTHOR]

Riccia sorocarpa Bisch., commonly known as common crystalwort, is a plant belonging to the Marchantiales order with a cosmopolitan distribution among a wide range of habitats: fields, gardens, waste ground, on paths, cliff tops, and thin soil over rocks or by water bodies. However, research into the genetic aspects of this species is limited. In this study, the chromosome-scale telomere-to-telomere genome of R. sorocarpa was assembled exclusively by Oxford Nanopore long-read sequencing and Pore-C technology. A high-quality chromosomal-scale assembly was obtained with a final genome size of 376.690 Mbp, contig N50 of 49.132 Mbp and 97.02% of the assembled contigs associated with the eight chromosomes. Genome assembly completeness was confirmed by BUSCO analysis accounting 91.8%. Among 27,626 total genes, 23,562 (85.29%) were functionally annotated. Moreover, collinearity of Marchantiales was analyzed as well as gene family evolution and DNA methylation profile. The availability of this genome, which is the second telomere-to-telomere liverwort assembly, opens up new avenues for in-depth analysis of R. sorocarpa genetic diversity and genomic characteristics. [ABSTRACT FROM AUTHOR]

Background : The rumen fluke, Calicophoron daubneyi, is the major paramphistome species infecting ruminants within Europe. Adult flukes reside within the rumen where they are in direct contact with a unique collection of microorganisms. Here, we report a 1.76-Gb draft genome for C. daubneyi, the first for any paramphistome species. Results: Several gene families have undergone specific expansion in C. daubneyi, including the peptidoglycan-recognition proteins (PGRPs) and DM9 domain-containing proteins, which function as pattern-recognition receptors, as well as the saposin-like proteins with putative antibacterial properties, and are upregulated upon arrival of the fluke in the microbe-rich rumen. We describe the first characterisation of a helminth PGRP and show that a recombinant C. daubneyi PGRP binds to the surface of bacteria, including obligate anaerobes from the rumen, via specific interaction with cell wall peptidoglycan. We reveal that C. daubneyi eggshell proteins lack L-DOPA typically required for eggshell crosslinking in trematodes and propose that C. daubneyi employs atypical eggshell crosslinking chemistry that produces eggs with greater stability. Finally, although extracellular digestion of rumen ciliates occurs within the C. daubneyi gut, unique ultrastructural and biochemical adaptations of the gastrodermal cells suggest that adult flukes also acquire nutrients via uptake of volatile fatty acids from rumen fluid. Conclusions: Our findings suggest that unique selective pressures, associated with inhabiting a host environment so rich in microbial diversity, have driven the evolution of molecular and morphological adaptations that enable C. daubneyi to defend itself against microorganisms, feed and reproduce within the rumen. [ABSTRACT FROM AUTHOR]

Background: Stress responses are key the survival of parasites and, consequently, also the evolutionary success of these organisms. Despite this importance, our understanding of the evolution of molecular pathways dealing with environmental stressors in parasitic animals remains limited. Here, we tested the link between adaptive evolution of parasite stress response genes and their ecological diversity and species richness. We comparatively investigated antioxidant, heat shock, osmoregulatory, and behaviour-related genes (foraging) in two model parasitic flatworm lineages with contrasting ecological diversity, Cichlidogyrus and Kapentagyrus (Platyhelminthes: Monopisthocotyla), through whole-genome sequencing of 11 species followed by in silico exon bait capture as well as phylogenetic and codon analyses. Results: We assembled the sequences of 48 stress-related genes and report the first foraging (For) gene orthologs in flatworms. We found duplications of heat shock (Hsp) and oxidative stress genes in Cichlidogyrus compared to Kapentagyrus. We also observed positive selection patterns in genes related to mitochondrial protein import (Hsp) and behaviour (For) in species of Cichlidogyrus infecting East African cichlids—a host lineage under adaptive radiation. These patterns are consistent with a potential adaptation linked to a co-radiation of these parasites and their hosts. Additionally, the absence of cytochrome P450 and kappa and sigma-class glutathione S-transferases in monogenean flatworms is reported, genes considered essential for metazoan life. Conclusions: This study potentially identifies the first molecular function linked to a flatworm radiation. Furthermore, the observed gene duplications and positive selection indicate the potentially important role of stress responses for the ecological adaptation of parasite species. [ABSTRACT FROM AUTHOR]

Ethylene is a signalling factor that plays a key role in the response of plants to abiotic stresses, such as cold stress. Recent studies have shown that the exogenous application of 1-aminocyclopropane-1-carboxylate (ACC), an ethylene promoter, affects plant cold tolerance. The cold-responsive specific gene DREB plays a crucial role in enhancing cold tolerance in plants by activating several cold-responsive (COR) genes. However, how the ethylene biosynthesis pathway regulates this gene in the cold response of thermophilic plants has yet to be fully elucidated. In this study, the thermophilic plant Plumbago indica L. was used as an example. Physiological experiments and transcriptomic analyses revealed that cold stress treatment induced the synthesis of endogenous ACC and regulated the ethylene signalling activator PiERF1, and cold signalling also activated PiDREB1A. Spray experiments also revealed that ACC-induced upregulation of the PiERF1 gene reduced the cold tolerance of P. indica and decreased the expression level of the PiDREB1A gene. These results indicate that ethylene signalling directly regulates the downstream gene PiERF1 and initiates the DREB‒COR cold-responsive signalling pathway to regulate cold tolerance, resulting in the negative regulation of cold tolerance in thermophilic plants. [ABSTRACT FROM AUTHOR]

Background: Plant mitochondrial genomes (mitogenomes) exhibit extensive structural variation yet extremely low nucleotide mutation rates, phenomena that remain only partially understood. The genus Gossypium, a globally important source of cotton, offers a wealth of long-read sequencing resources to explore mitogenome and plastome variation and dynamics accompanying the evolutionary divergence of its approximately 50 diploid and allopolyploid species. Results: Here, we assembled 19 mitogenomes from Gossypium species, representing all genome groups (diploids A through G, K, and the allopolyploids AD) based on a uniformly applied strategy. A graph-based mitogenome assembly method revealed more alternative structural conformations than previously recognized, some of which confirmed the mitogenome structure reported in earlier studies on cotton. Using long-read data, we quantified alternative conformations mediated by recombination events between repeats, and phylogenetically informative structural variants were noted. Nucleotide substitution rate comparisons between coding and non-coding regions revealed low mutation rates across the entire mitogenome. Genome-wide mapping of nuclear organellar DNA transfers (NUOTs) in Gossypium revealed a nonrandom distribution of transfers in the nuclear genome. In cotton, the fate of NUOT events varied, with mitochondrion-to-nucleus transfer (NUMT) predominantly retained as short fragments in the nuclear genome, with more plastid sequences integrated into the nucleus. Phylogenetic relationships inferred using different data sets highlighted distinct evolutionary histories among these cellular compartments, providing ancillary evidence relevant to the evolutionary history of Gossypium. Conclusions: A comprehensive analysis of organellar genome variation demonstrates complex structural variation and low mutation rates across the entire mitogenome and reveals the history of organellar genome transfer among the three genomes throughout the cotton genus. The findings enhance our general understanding of mitogenome evolution, comparative organellar and nuclear evolutionary rates, and the history of inter-compartment genomic integration. [ABSTRACT FROM AUTHOR]

Argali stands as the largest species among wild sheep in Central and East Asia, with a concerning rate of decline estimated at 30%. The intraspecific taxonomy of argali remains contentious due to limited genomic data and unclear geographic separation. In this study, we constructed a chromosome-level genome assembly and annotation for the Tibetan argali (O. a. hodgsoni), together with population genomic resequencing of 32 individuals representing four subspecies. The contig-level genome was 2.64 Gb in size, with a contig N50 length of 71.69 Mb and an estimated genomic completeness of 96.01%. Using Hi-C sequencing data scaffolding, 99.90% of initially assembled sequences were mapped and oriented onto 28 pseudo-chromosomes except the Y chromosome. Annotation uncovered 21,564 protein-coding genes and 46.38% repeat sequences. The average coverage of the population resequencing data was 23.74 with mean mapping ratio up to of 97.19%. The high-quality genome assembly and annotation of the Tibetan argali, coupled with the high-depth population genomic data, will serve as a valuable genetic resource for studies on the taxonomy and conservation of argali. [ABSTRACT FROM AUTHOR]

Three-spotted seahorse (Hippocampi trimaculata) is a unique fish with important economic and medicinal values, and its total chromosome number is potentially quite different from other seahorse species. Herein, we constructed a chromosome-level genome assembly for this special seahorse by integration of MGI short-read, PacBio HiFi long-read and Hi-C sequencing techniques. A 416.57-Mb haplotypic genome assembly was obtained. Subsequently, 99.38% of its scaffold sequences were anchored onto 18 chromosomes, with identification of 29.1% repeat sequences in the assembled genome. Additional karyotype analysis validated the diploid chromosomes of 2n = 36, which are remarkably different from other seahorses' 2n = 42 or 44. The genome completeness (BUSCO score: 96.5%, CEGMA score: 97.87%) confirmed that this chromosome-scale assembly is indeed of high quality. Moreover, a total of 18,712 protein-coding genes were annotated, of which 96.36% could be predicted with functions. Based on construction of a phylogenetic tree, we estimated that Hippocampus and Syngnathoides diverged approximately 50.1 million years ago (Mya). Taken together, our genome data presented in this study provide a valuable genetic resource for numerical chromosome changes and in-depth evolutionary and functional investigations, as well as conservation and molecular breeding of this endangered teleost. [ABSTRACT FROM AUTHOR]

Megachile sculpturalis Smith, 1853 native to East Asia, is an important solitary bee species that has invaded both Europe and the United States. This study provides the first chromosome-level genome assembly of M. sculpturalis using a combination of Nanopore long reads, Illumina short reads, and Hi-C data. The genome comprises 296.99 Mb distributed across 16 chromosomes. N50, L50 and BUSCO completeness reached 19.128 Mb, 7 scaffolds, and 96.7%, respectively. The genome contains 104 Mb repetitive elements (35.02% of the assembly size) and 11,446 predicted protein-coding genes. This chromosome-level genome will serve as an essential genomic resource for future research on Megachilidae. [ABSTRACT FROM AUTHOR]

Horizontal transfer of genetic material in eukaryotes has rarely been documented over short evolutionary timescales. Here, we show that two retrotransposons, Shellder and Spoink, invaded the genomes of multiple species of the melanogaster subgroup within the last 50 years. Through horizontal transfer, Spoink spread in D. melanogaster during the 1980s, while both Shellder and Spoink invaded D. simulans in the 1990s. Possibly following hybridization, D. simulans infected the island endemic species D. mauritiana (Mauritius) and D. sechellia (Seychelles) with both TEs after 1995. In the same approximate time-frame, Shellder also invaded D. teissieri, a species confined to sub-Saharan Africa. We find that the donors of Shellder and Spoink are likely American Drosophila species from the willistoni, cardini, and repleta groups. Thus, the described cascade of TE invasions could only become feasible after D. melanogaster and D. simulans extended their distributions into the Americas 200 years ago, likely aided by human activity. Our work reveals that cascades of TE invasions, likely initiated by human-mediated range expansions, could have an impact on the genomic and phenotypic evolution of geographically dispersed species. Within a few decades, TEs could invade many species, including island endemics, with distributions very distant from the donor of the TE. Horizontal transfer of genetic material in eukaryotes has rarely been documented over short evolutionary timescales. Here, the authors show that two transposable elements, Shellder and Spoink, invaded the genomes of multiple Drosophila species within the last 50 years. [ABSTRACT FROM AUTHOR]

Genomics is an increasingly important part of biology research. However, educating undergraduates in genomics is not yet a standard part of life sciences curricula. We believe this is, in part, due to a lack of standard concepts for the teaching of genomics. To address this deficit, the members of the Genomics Education Alliance created a set of genomics concepts that was then further refined by input from a community of undergraduate educators who engage in genomics instruction. The final genomics concepts list was compared to existing learning frameworks, including the Vision and Change initiative (V&C), as well as ones for genetics, biochemistry and molecular biology, and bioinformatics. Our results demonstrate that the new genomics framework fills a niche not addressed by previous inventories. This new framework should be useful to educators seeking to design stand-alone courses in genomics as well as those seeking to incorporate genomics into existing coursework. Future work will involve designing curriculum and assessments to go along with this genomics learning framework. [ABSTRACT FROM AUTHOR]

Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac-derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties of piggyBat from Myotis lucifugus, the only known active mammalian DNA transposon, and show that its low activity in human cells is due to subterminal inhibitory DNA sequences. Activity can be dramatically improved by their removal, suggesting the existence of a mechanism for the suppression of transposon activity. The cryo-electron microscopy structure of the piggyBat transposase pre-synaptic complex showed an unexpected mode of DNA binding and recognition using C-terminal domains that are topologically different from those of the piggyBac transposase. Here we show that structure-based rational re-engineering of the transposase through the removal of putative phosphorylation sites and a changed domain organization - in combination with truncated transposon ends - results in a transposition system that is at least 100-fold more active than wild-type piggyBat. piggyBat is the only known DNA transposon active in mammals. Here the authors used cellular assays and 3D structure to show that sequences within the transposon ends restrict activity, and also how transposition activity can be substantially increased. [ABSTRACT FROM AUTHOR]

Background: Ortholog prediction, essential for various genomic research areas, faces growing inconsistencies amidst the expanding array of ortholog databases. The common strategy of computing consensus orthologs introduces additional arbitrariness, emphasizing the need to examine the causes of such inconsistencies and identify proteins susceptible to prediction errors. Results: We introduce the Signal Jaccard Index (SJI), a novel metric rooted in unsupervised genome context clustering, designed to assess protein similarity. Leveraging SJI, we construct a protein network and reveal that peripheral proteins within the network are the primary contributors to inconsistencies in orthology predictions. Furthermore, we show that a protein's degree centrality in the network serves as a strong predictor of its reliability in consensus sets. Conclusions: We present an objective, unsupervised SJI-based network encompassing all proteins, in which its topological features elucidate ortholog prediction inconsistencies. The degree centrality (DC) effectively identifies error-prone orthology assignments without relying on arbitrary parameters. Notably, DC is stable, unaffected by species selection, and well-suited for ortholog benchmarking. This approach transcends the limitations of universal thresholds, offering a robust and quantitative framework to explore protein evolution and functional relationships. [ABSTRACT FROM AUTHOR]

The flat-headed loach (Oreonectes platycephalus) is a small fish inhabiting headwaters of hillstreams of southern China. Its local populations are characterized by low genetic diversity and exceptionally high differentiation, making it an ideal model for studying small population isolates' persistence and adaptive potential. However, the lack of Oreonectes reference genomes limits endeavours toward these ambitions. We assembled the first haplotype-resolved chromosome-level genome of the genus Oreonectes using PacBio HiFi and Hi-C technologies. This genome consists of two haplotypes (24 pseudo-chromosomes in each), with sizes of 565.68 Mb (haplotype A) and 521.13 Mb (haplotype B) and scaffold N50 lengths of 22.80 Mb and 21.91 Mb, respectively. Chr01 was identified as the likely sex chromosome pair. After masking repetitive elements which accounted for 34.43% to 36.44% of the genome, there are 27,127 protein-coding genes in haplotype A and 25,576 in haplotype B. The availability of this haplotype-resolved chromosome-level reference genome will facilitate the study of population and conservation genetics of the flat-headed loach and other Oreonectes species. [ABSTRACT FROM AUTHOR]

The sunflower (Helianthus annuus L.), belonging to the Asteraceae family, is the world's fourth most important oil crop. Sunflower cultivars are categorized into oilseed and confectionery types. Here, we present chromosome-level genome assemblies of two Chinese sunflower cultivars-oilseed and confectionery-using PacBio HiFi and Hi-C sequencing. The oilseed cultivar, OXS, has a genome assembly spanning 3.03 Gb with 99.58% of sequences anchored to 17 chromosomes and a contig N50 length of 154.78 Mb. The first published confectionery cultivar genome, YDS, mirrors this closely with a 3.02 Gb assembly, contig N50 length of 153.87 Mb and 99.40% of sequences mapped similarly. Gene completeness reached 98.2% for OXS and 98.4% for YDS, with LTR Assembly Index scores of 24.73 and 25.85, respectively. Comparative genomics identified rapidly evolving gene families linked to synthesis, growth, and stress defense. Additionally, we found high collinearity between the YDS and OXS genomes, despite three significant inversions, and detected 15,056 large deletions and insertions. These findings lay a robust foundation for advanced genomic research and breeding innovations in sunflowers. [ABSTRACT FROM AUTHOR]

Tigecycline is a last-resort antibiotic to treat complicated infections caused by multidrug-resistant pathogens, while the emergence of plasmid-mediated tet(X) family severely compromises its clinical efficacy. Novel antimicrobial strategies not limited to new antibiotics in pharmaceutical pipeline are urgently needed. Herein, we reveal the metabolic disparities between tet(X)-negative and -positive E. coli, including distinct energy demand patterns under tigecycline exposure. In particular, the cysteine and methionine metabolism pathway is remarkably downregulated in tet(X)-positive bacteria. More importantly, we find that the addition of exogenous L-methionine (Met) effectively resensitizes tet(X)-positive pathogens to tigecycline. Our mechanistic analysis demonstrates that exogenous Met promotes intracellular tigecycline accumulation by upregulating bacterial proton motive force. Moreover, Met accelerates the conversion to S-adenosyl-L-methionine, an essential methyl donor, thereby enhancing 5mC methylation modification in the promoter region of tet(X4) gene and reducing its expression. Consistently, the potentiation of Met to tigecycline is abolished in tet(X4)-carrying E. coli Δdcm but restored in dcm-complementary bacteria, which encodes DNA-cytosine methyltransferase. In multiple animal models of infection, Met markedly potentiates the effectiveness of tigecycline against pathogenic E. coli and K. pneumoniae. Overall, this work highlights the therapeutic potential of Met in overcoming plasmid-mediated high-level tigecycline resistance, and provides a new paradigm to enhance antibiotic efficacy by harnessing cellular metabolic networks as well as epigenetic modifications. The dissemination of plasmid-encoded tigecycline resistance gene tet(X) poses a global threat to public health, necessitating the development of innovative strategies. Here, the authors reveal that the supplementation of L-methionine not only facilitates intracellular accumulation of tigecycline but also reduces tet(X) expression by augmenting the 5mC methylation modification in its promoter region, thereby overcoming plasmid-mediated high-level tigecycline resistance. [ABSTRACT FROM AUTHOR]

Objective: Breast cancer is a widely prevalent and life-threatening malignancy that affects women worldwide. The identification of novel molecular markers associated with tumor progression is highly important for enhancing early detection, tailoring treatment approaches, and monitoring therapeutic outcomes. In this study, we investigated the expression patterns of four long noncoding RNAs (lncRNAs): USP30 antisense RNA1 (USP30-AS1), ELFN1 antisense RNA1 (ELFN1-AS1), GAS8 antisense RNA1 (GAS8-AS1), and small nucleolar RNA host gene 11 (SNHG11). Results: In breast cancer specimens, USP30-AS1 and GAS8-AS1 expression was decreased, whereas ELFN1-AS1 and SNHG11 expression was increased in breast cancer tissues compared with adjacent noncancer tissues. Decreased USP30-AS1 levels were associated with a smaller tumor size, lower tumor grade and stage, and the absence of lymphatic and vascular invasion. Lower GAS8-AS1 expression was associated with a lower tumor grade and positive estrogen and progestin receptor status. Elevated ELFN1-AS1 expression was associated with breast cancer that lacked P53 mutation. These changes suggest their promise as biomarkers for distinguishing between cancerous and noncancerous tissues. [ABSTRACT FROM AUTHOR]

Drought is one of the main environmental factors affecting plant survival and growth. Atraphaxis bracteata is a common desert plant mainly utilized in afforestation and desertification control. This study analyzed the morphological, physiological and molecular regulatory characteristics of different organs of A. bracteata under drought stress. The results showed that with the intensification of drought stress, the height, root length and leaf area of A. bracteata seedlings decreased, while the content of osmotic substances and antioxidant enzyme activity increased. Furthermore, a total of 63 907 non-redundant transcript sequences, of which 55 574 transcripts were annotated, 248 178 FLNC sequences, 107 112 high-quality consistent sequences and 291 314 CCSs were obtained from Iso-Seq. Meanwhile, a total of 2 039 AS events, 22 919 SSR, 40 404 CDS and 5 902 lncRNA were also obtained. The RNA-Seq analysis results revealed that a total of 2 821, 3 907 and 5 532 DETs were identified from roots, stems and leaves, respectively, and which had significantly enrichment in "circadian rhythm-plant" and "starch and sucrose metabolism" pathway. These results would be great significance for further research on the stress resistance of A. bracteata and these DETs function. [ABSTRACT FROM AUTHOR]

Transfer RNAs (tRNAs) are noncoding RNAs involved in protein biosynthesis and have noncanonical roles in cellular metabolism, such as RNA silencing and the generation of transposable elements. Extensive tRNA gene duplications, modifications to mature tRNAs, and complex secondary and tertiary structures impede tRNA sequencing. As such, a comparative genomic analysis of complete tRNA sets is an alternative to understanding the evolutionary processes that gave rise to the extant tRNA sets. Although the tRNA gene (tDNA) structure and distribution in prokaryotes and eukaryotes, specifically in vertebrates, yeasts, and flies, are well understood, there is little information regarding plants. A detailed and comprehensive analysis and annotation of tDNAs from the genomes of 44 eudicots, 20 monocots, and five other non-eudicot and non-monocot species belonging to the Ceratophyllaceae and the ANA (Amborellales, Nymphaeales, and Austrobaileyales) clade will provide a global picture of plant tDNA structure and organization. Plant genomes exhibit varying numbers of nuclear tDNAs, with only the monocots showing a strong correlation between nuclear tDNA numbers and genome sizes. In contrast, organellar tDNA numbers varied little among the different lineages. A high degree of tDNA duplication in eudicots was detected, whereby most eudicot nuclear genomes (91%) and only a modest percentage of monocot (65%) and ANA nuclear genomes (25%) contained at least one tDNA cluster. Clusters of tRNATyr–tRNASer and tRNAIle genes were found in eudicot and monocot genomes, respectively, while both eudicot and monocot genomes showed clusters of tRNAPro genes. All plant genomes had intron-containing tRNAeMet and tRNATyr genes with modest sequence conservation and a strictly conserved tRNAAla-AGC species. Regulatory elements found upstream (TATA-box and CAA motifs) and downstream (poly(T) signals) of the tDNAs were present in only a fraction of the detected tDNAs. A and B boxes within the tDNA coding region show varying consensus sequences depending on the tRNA isotype and lineage. The chloroplast genomes, but not the mitogenomes, possess relatively conserved tRNA gene organization. These findings reveal differences and patterns acquired by plant genomes throughout evolution and can serve as a foundation for further studies on plant tRNA gene function and regulation. [ABSTRACT FROM AUTHOR]

Introduction: Long non-coding RNAs (lncRNAs) are a fascinating, but still largely uncharacterized, class of genes. Recently, lncRNAs have attracted significant attention due to their emerging functions in development and disease. The role of lncRNAs in chromosome instability or aneuploidy is not extensively studied. Methods: We started with the objective of characterizing lncRNAs that play an important role in chromosome instability (CIN) or aneuploidy. Here, we report the initial functional characterization of PURPL in the context of chromosomal instability or aneuploidy. Results: We report the over-expression of lncRNA PURPL in three experimental models of chromosomal instability, or aneuploidy. In addition, the study also showed that the extent or magnitude of PURPL expression is dependent upon p53 status. Our research also showed that turning off PURPL is enough to create a CIN phenotype in RPE-1 cell lines that were previously karyotypically stable. Moreover, PURPL knockdown cells are more sensitive to CIN or aneuploidy inducers. Discussion: These findings show that PURPL is essential for maintaining chromosomal or genomic stability in mammalian cells. Collectively, the study demonstrated that lncRNA-PURPL significantly contributes to CIN, or aneuploidy. [ABSTRACT FROM AUTHOR]

In the era of biodiversity genomics, it is crucial to ensure that annotations of protein-coding gene repertoires are accurate. State-of-the-art tools to assess genome annotations measure the completeness of a gene repertoire but are blind to other errors, such as gene overprediction or contamination. We introduce OMArk, a software package that relies on fast, alignment-free sequence comparisons between a query proteome and precomputed gene families across the tree of life. OMArk assesses not only the completeness but also the consistency of the gene repertoire as a whole relative to closely related species and reports likely contamination events. Analysis of 1,805 UniProt Eukaryotic Reference Proteomes with OMArk demonstrated strong evidence of contamination in 73 proteomes and identified error propagation in avian gene annotation resulting from the use of a fragmented zebra finch proteome as a reference. This study illustrates the importance of comparing and prioritizing proteomes based on their quality measures. A new tool checks the quality of gene annotations in genome sequences. [ABSTRACT FROM AUTHOR]

Current approaches for inserting autonomous transgenes into the genome, such as CRISPR–Cas9 or virus-based strategies, have limitations including low efficiency and high risk of untargeted genome mutagenesis. Here, we describe precise RNA-mediated insertion of transgenes (PRINT), an approach for site-specifically primed reverse transcription that directs transgene synthesis directly into the genome at a multicopy safe-harbor locus. PRINT uses delivery of two in vitro transcribed RNAs: messenger RNA encoding avian R2 retroelement-protein and template RNA encoding a transgene of length validated up to 4 kb. The R2 protein coordinately recognizes the target site, nicks one strand at a precise location and primes complementary DNA synthesis for stable transgene insertion. With a cultured human primary cell line, over 50% of cells can gain several 2 kb transgenes, of which more than 50% are full-length. PRINT advantages include no extragenomic DNA, limiting risk of deleterious mutagenesis and innate immune responses, and the relatively low cost, rapid production and scalability of RNA-only delivery. Transgenes are inserted into human cells by 2-RNA delivery of a retroelement protein and template. [ABSTRACT FROM AUTHOR]

Background: CRISPRi screening has become a powerful approach for functional genomic research. However, the off-target effects resulting from the mismatch tolerance between sgRNAs and their intended targets is a primary concern in CRISPRi applications. Results: We introduce Guide Library Designer (GLiDe), a web-based tool specifically created for the genome-scale design of sgRNA libraries tailored for CRISPRi screening in prokaryotic organisms. GLiDe incorporates a robust quality control framework, rooted in prior experimental knowledge, ensuring the accurate identification of off-target hits. It boasts an extensive built-in database, encompassing 1,397 common prokaryotic species as a comprehensive design resource. It also provides the capability to design sgRNAs for newly discovered organisms by accepting uploaded design resource. We further demonstrated that GLiDe exhibits enhanced precision in identifying off-target binding sites for the CRISPRi system. Conclusions: We present a web server that allows the construction of genome-scale CRISPRi sgRNA libraries for prokaryotes. It mitigates off-target effects through a robust quality control framework, leveraging prior experimental knowledge within an end-to-end, user-friendly pipeline. [ABSTRACT FROM AUTHOR]

Legionella pneumophila utilizes the Dot/Icm type IVB secretion system to deliver hundreds of effector proteins inside eukaryotic cells to ensure intracellular replication. Our understanding of the molecular functions of the largest pathogenic arsenal known to the bacterial world remains incomplete. By leveraging advancements in 3D protein structure prediction, we provide a comprehensive structural analysis of 368 L. pneumophila effectors, representing a global atlas of predicted functional domains summarized in a database (https://pathogens3d.org/legionella-pneumophila). Our analysis identified 157 types of diverse functional domains in 287 effectors, including 159 effectors with no prior functional annotations. Furthermore, we identified 35 cryptic domains in 30 effector models that have no similarity with experimentally structurally characterized proteins, thus, hinting at novel functionalities. Using this analysis, we demonstrate the activity of thirteen functional domains, including three cryptic domains, predicted in L. pneumophila effectors to cause growth defects in the Saccharomyces cerevisiae model system. This illustrates an emerging strategy of exploring synergies between predictions and targeted experimental approaches in elucidating novel effector activities involved in infection. Synopsis: Legionella pneumophila encodes the largest arsenal of eukaryotic host-manipulating proteins, called effectors. Leveraging high-throughput structural prediction tools, coupled with in cellulo assays, revealed previously unrecognized functional and cryptic domains in L. pneumophila effectors. Analysis revealed over 150 structurally diverse domains in 287 effectors, reflective of the diverse mechanisms required to modulate L. pneumophila's eukaryotic hosts ranging from protozoa to human alveolar macrophages. Over half of the predicted functional domains are present in only one effector, while cysteine proteases and kinases are the most prominent domain categories. An even larger presence of so-called tandem repeat structural motifs in effector proteins is suggested to facilitate protein-protein interactions. Over 30 identified domains, some of which are present in other pathogens, showed no structural similarity with characterized proteins, suggestive of novel biochemical activities. Legionella pneumophila encodes the largest arsenal of eukaryotic host-manipulating proteins, called effectors. Leveraging high-throughput structural prediction tools, coupled with in cellulo assays, revealed previously unrecognized functional and cryptic domains in L. pneumophila effectors. [ABSTRACT FROM AUTHOR]

Microsatellite markers are cost-effective, rapid, efficient, and show great advantages in in large-sample kinship analysis and population structure studies. However, microsatellite loci are seriously underdeveloped in non-model organisms. The plateau zokor (Eospalax baileyi) is a key species living underground in the Tibetan Plateau, the effective management of which has long been challenging. In this study, we analyzed the distribution characteristics and functions of microsatellites in the genome of plateau zokors, and their polymorphic sites. The mononucleotide and dinucleotide types being the most abundant in the genome. The largest number of microsatellites and their abundance in the intergenic region whereas the smallest number of microsatellites and their abundance in the coding region. The coding sequences containing microsatellites were annotated to 52 major functional genes and assigned 19,358 Gene Ontology entries. The Kyoto Encyclopedia of Genes and Genomes pathway was the most enriched in the signal transduction pathway. Thirteen pairs of polymorphic loci were successfully amplified, with the number of alleles ranging from 3 to 8, observed heterozygosity ranging from 0.059 to 0.810, and expected heterozygosity ranging from 0.469 to 0.854. These microsatellite markers provide a cornerstone for studies on the identification of parentage and population genetics of plateau zokors. [ABSTRACT FROM AUTHOR]

Promoters are essential DNA sequences that initiate transcription and regulate gene expression. Precisely identifying promoter sites is crucial for deciphering gene expression patterns and the roles of gene regulatory networks. Recent advancements in bioinformatics have leveraged deep learning and natural language processing (NLP) to enhance promoter prediction accuracy. Techniques such as convolutional neural networks (CNNs), long short-term memory (LSTM) networks, and BERT models have been particularly impactful. However, current approaches often rely on arbitrary DNA sequence segmentation during BERT pre-training, which may not yield optimal results. To overcome this limitation, this article introduces a novel DNA sequence segmentation method. This approach develops a more refined dictionary for DNA sequences, utilizes it for BERT pre-training, and employs an Inception neural network as the foundational model. This BERT-Inception architecture captures information across multiple granularities. Experimental results show that the model improves the performance of several downstream tasks and introduces deep learning interpretability, providing new perspectives for interpreting and understanding DNA sequence information. The detailed source code is available at https://github.com/katouMegumiH/Promoter_BERT. [ABSTRACT FROM AUTHOR]

Abstract Background The increasing amount of genomic data calls for tools that can create genome-scale phylogenies quickly and efficiently. Existing tools rely on large reference databases or require lengthy de novo calculations to identify orthologues, meaning that they have long run times and are limited in their taxonomic scope. To address this, we created getphylo, a python tool for the rapid generation of phylogenetic trees de novo from annotated sequences. Results We present getphylo (Genbank to Phylogeny), a tool that automatically builds phylogenetic trees from annotated genomes alone. Orthologues are identified heuristically by searching for singletons (single copy genes) across all input genomes and the phylogeny is inferred from a concatenated alignment of all coding sequences by maximum likelihood. We performed a thorough benchmarking of getphylo against two existing tools, autoMLST and GTDB-tk, to show that it can produce trees of comparable quality in a fraction of the time. We also demonstrate the flexibility of getphylo across four case studies including bacterial and eukaryotic genomes, and biosynthetic gene clusters. Conclusions getphylo is a quick and reliable tool for the automated generation of genome-scale phylogenetic trees. getphylo can produce phylogenies comparable to other software in a fraction of the time, without the need large local databases or intense computation. getphylo can rapidly identify orthologues from a wide variety of datasets regardless of taxonomic or genomic scope. The usability, speed, flexibility of getphylo makes it a valuable addition to the phylogenetics toolkit.

Abstract Current temporal studies of DNA replication are either low-resolution or require complex cell synchronisation and/or sorting procedures. Here we introduce Nanotiming, a single-molecule, nanopore sequencing-based method producing high-resolution, telomere-to-telomere replication timing (RT) profiles of eukaryotic genomes by interrogating changes in intracellular dTTP concentration during S phase through competition with its analogue bromodeoxyuridine triphosphate (BrdUTP) for incorporation into replicating DNA. This solely demands the labelling of asynchronously growing cells with an innocuous dose of BrdU during one doubling time followed by BrdU quantification along nanopore reads. We demonstrate in S. cerevisiae model eukaryote that Nanotiming reproduces RT profiles generated by reference methods both in wild-type and mutant cells inactivated for known RT determinants. Nanotiming is simple, accurate, inexpensive, amenable to large-scale analyses, and has the unique ability to access RT of individual telomeres, revealing that Rif1 iconic telomere regulator selectively delays replication of telomeres associated with specific subtelomeric elements.

Abstract 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.

Transposable elements (TEs) are significant drivers of genome evolution, yet their recent dynamics and impacts within and among species, as well as the roles of host genes and non-coding RNAs in the transposition process, remain elusive. With advancements in large-scale pan-genome sequencing and the development of open data sharing, large-scale comparative genomics studies have become feasible. Here, we performed complete de novo TE annotations and identified active TEs in 310 plant genome assemblies across 119 species and seven crop populations. Using 811 high-quality genomes, we detected 13 844 553 TE-induced structural variants (TE-SVs), providing unprecedented resolution in delineating recent TE activities. Our integrative analysis revealed a mutual evolutionary relationship between TEs and host genomes. On one hand, host genes and ncRNAs are involved in the transposition process, as evidenced by their colocalization and coactivation with TEs, and may play a role in chromatin regulation. On the other hand, TEs drive genetic innovation by promoting the duplication of host genes and inserting into regulatory regions. Moreover, genes influenced by active TEs are linked to plant growth, nutrient absorption, storage metabolism and environmental adaptation, aiding in crop domestication and adaptation. This TE dynamics atlas not only reveals evolutionary and functional features linked to transposition activity but also highlights the role of TEs in crop domestication and adaptation, paving the way for future exploration of TE-mediated genome evolution and crop improvement strategies., (© 2025 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Ongoing efforts to improve sheep reference genome assemblies still leave many gaps and incomplete regions, resulting in a few common failures and errors in genomic studies. Here, we report a 2.85-Gb gap-free telomere-to-telomere genome of a ram (T2T-sheep1.0), including all autosomes and the X and Y chromosomes. This genome adds 220.05 Mb of previously unresolved regions and 754 new genes to the most updated reference assembly ARS-UI_Ramb_v3.0; it contains four types of repeat units (SatI, SatII, SatIII and CenY) in centromeric regions. T2T-sheep1.0 has a base accuracy of more than 99.999%, corrects several structural errors in previous reference assemblies and improves structural variant detection in repetitive sequences. Alignment of whole-genome short-read sequences of global domestic and wild sheep against T2T-sheep1.0 identifies 2,664,979 new single-nucleotide polymorphisms in previously unresolved regions, which improves the population genetic analyses and detection of selective signals for domestication (for example, ABCC4) and wool fineness (for example, FOXQ1)., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The publication of several high-quality genomes has contributed greatly to clarifying the evolution of citrus. However, due to their complex genetic backgrounds, the origins and evolution of many citrus species remain unclear. We assembled de novo the 294-Mbp chromosome-level genome of a more than 200-year-old primitive papeda (DYC002). Comparison between the two sets of homologous chromosomes of the haplotype-resolved genome revealed 1.2% intragenomic variations, including 1.75 million SNPs, 149,471 insertions and 154,215 deletions. Using this genome as a reference, we resequenced and performed population and phylogenetic analyses of 378 representative citrus accessions. Our study confirmed that the primary origin center of core Citrus species is in South China, particularly in the Himalaya-Hengduan Mountains. Papeda species are an ancient Citrus type compared with C. ichangensis. We found that the evolution of the Citrus genus followed two radiations through two routes (to East China and Southeast Asia) along river systems. Evidence for the origin and evolution of some individual citrus species was provided. Papeda probably played an important role in the origins of Australian finger lime, citrons, Honghe papeda and pummelos; Ichang papeda originated from Yuanjiang city of Yunnan Province, China, and C. mangshanensis has a close relationship with kumquat and Ichang papeda. Moreover, the Hunan and Guangdong Provinces of China are predicted to be the origin center of mandarin, sweet orange and sour orange. Additionally, our study revealed that fruit bitterness was significantly selected against during citrus domestication. Taken together, this study provides new insight into the origin and evolution of citrus species and may serve as a valuable genomic resource for citrus breeding and improvement., (© 2025 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

The shift to reductionist biology at the dawn of the genome era yielded a 'parts list' of plant genes and a nascent understanding of complex biological processes. Today, with the genomics era in full swing, advances in high-definition genomics enabled precise temporal and spatial analyses of biological systems down to the single-cell level. These insights, coupled with artificial intelligence-driven in silico design, are propelling the development of the first synthetic plants. By integrating reductionist and systems approaches, researchers are not only reimagining plants as sources of food, fiber, and fuel but also as 'environmental thermostats' capable of mitigating the impacts of a changing climate., (© 2025 The Author(s). New Phytologist © 2025 New Phytologist Foundation.)