Search Results (83)

Timeless and its fission yeast ortholog Swi1 are evolutionarily conserved components of the replication fork protection complex that ensures faithful DNA replication and genome stability. While their nuclear roles are well-characterized, their roles in mitochondrial genome maintenance remain unknown. Here, we demonstrate a previously unrecognized connection between Timeless/Swi1 and mitochondrial homeostasis. In fission yeast, swi1 deletion increased association of the DNA repair protein Rad52 with mitochondrial DNA sequences across the mitochondrial genome, suggesting altered mitochondrial genome maintenance. Unexpectedly, swi1∆ cells showed an increased mtDNA copy number and improved growth under respiratory conditions, suggesting activation of compensatory mechanisms that promote mitochondrial genome maintenance. The loss of Swi1 also partially rescued the growth defect under respiratory conditions and mtDNA loss associated with depletion of mitochondrial DNA polymerase γ, linking Swi1 to pathways regulating mitochondrial replication under stress. Consistent with these phenotypes, transcriptomic and pathway enrichment analyses revealed transcriptional changes indicative of reduced glycolysis and enhanced oxidative phosphorylation, suggesting a shift toward respiratory metabolism. In human cells, Timeless depletion elicited distinct mitochondrial responses depending on the cell type. While Timeless-depleted TE-11 and Saos-2 cells elicited mitochondrial phenotypes comparable to those observed in fission yeast, Timeless depletion in U-2 OS cells led to reduced mtDNA copy number, elevated mitochondrial reactive oxygen species, and decreased mitochondrial membrane potential and mass, consistent with mitochondrial dysfunction. Despite these phenotypic differences, both fission yeast and human cells exhibited elevated levels of orthologs of the mitochondrial transcription factor A (TFAM) and the oxidative stress regulator NRF2, suggesting the conserved activation of compensatory mitochondrial and antioxidant pathways. Together, these findings identify an evolutionarily conserved connection between Timeless/Swi1 and mitochondrial homeostasis and reveal distinct adaptive responses to mitochondrial stress. Full article

Background/Objectives: The long-spined sea urchin Diadema antillarum is a keystone herbivore whose grazing maintains Caribbean coral reefs; basin-wide mass mortalities in 1983–1984 and 2022 have made genomic resources a conservation priority, yet no nuclear genome existed for the species. We aimed to generate the first nuclear reference and to resolve the high heterozygosity that complicates genome assembly in broadcast-spawning marine invertebrates. Methods: For the assembly, we combined PacBio HiFi, Oxford Nanopore, and Illumina sequencing. Genome size and heterozygosity were estimated by k-mer profiling. We compared standard and haplotype-aware assembly strategies (hifiasm), evaluated completeness with BUSCO, and annotated repeats using a species-specific RepeatModeler library. Results: k-mer profiling estimated a haploid genome of ~703 Mb with 2.52% heterozygosity. Standard assembly then produced an inflated 1.75 Gb assembly (98.4% BUSCO-complete but 84.4% duplicated), indicating retention of both haplotypes. Haplotype-aware reassembly separated this into a collapsed primary assembly (1.03 Gb) and two phased haplotypes (0.95 and 0.89 Gb), each comparable in size to the chromosome-level congener D. antillarum (886 Mb). BUSCO completeness reached 99.0%, with single-copy orthologs rising to 85–90%, and reference-free consensus quality reached QV 44.5 (Merqury; initial assembly). This genome is repeat-rich (42.84% repetitive; 29.96% unclassified). Conclusions: We provide the collapsed primary assembly together with both phased haplotypes as a haplotype-resolved reference for D. antillarum, establishing a foundation for immunogenomic, comparative, and population-genetic studies and for monitoring and restoration of this ecologically critical species. More broadly, the study shows that haplotype-aware assembly is essential for resolving such highly heterozygous genomes and delivers the genomic foundation needed to guide the conservation of this keystone Caribbean reef species. Full article

The reliable identification of cultivated and naturalised mulberries is complicated by morphological plasticity, the historical movement of the planting material, and partly discordant nuclear and plastid signals. We evaluated four field-identified Morus alba L. trees, one from each of four localities in Mangystau, Western Kazakhstan, using archived consensus sequences for ITS, matK, rbcL, and trnH-psbA, together with a low-coverage Oxford Nanopore Technologies (ONT) dataset. Because the design comprised one tree per locality (n = 4), analyses were restricted to accession-level descriptive comparisons, and no population-genetic, phylogeographic, or formal phylogenetic inference was attempted. Archived alignment summaries indicated mean pairwise distances of 0.13% for matK, 0.44% for ITS, 0.94% for rbcL, and 2.92% for trnH-psbA; these values are reported as retained dataset descriptors rather than estimates of population diversity. An additional product generated with Rosaceae-derived s6pdh primers was excluded because the target identity and orthology could not be verified. The ONT run yielded 19,958 pass reads (69.27 Mb; read N50 3574 bp). Reference-enriched assembly produced a 14,824 bp candidate plastid-associated contig, approximately 9.3% of a typical Morus plastome. Its short length, incomplete and non-collinear annotations, and the absence of retained depth, polishing, assembly graph, and join support diagnostics preclude its interpretation as a complete, circular, or structurally validated plastome. This study provides a transparent pilot baseline for Mangystau mulberries and establishes quality control criteria for replicated sampling, validated markers, and deeper organelle sequencing. Full article

Mitochondrial genomes are widely used in insect taxonomy and phylogenetics, but their signals may conflict with morphology and nuclear genomic evidence because the mitochondrial genome represents a single maternally inherited locus. Here, we assembled complete mitochondrial genomes of four pygmy grasshoppers, Zhengitettix transpicula, Formosatettix sp., Gibbotettix parvipulvillus, and Bolivaritettix sp., using PacBio HiFi reads. The four mitogenomes ranged from 15,152 to 17,976 bp and contained the typical 37 mitochondrial genes. Mitochondrial phylogenies inferred by maximum likelihood and Bayesian methods were topologically identical and recovered several well-supported tetrigid relationships, including a close relationship between Formosatettix sp. and Bolivaritettix sp. However, Z. transpicula was unexpectedly placed near Macromotettixoides rather than close to other Zhengitettix representatives. In contrast, a morphology-based tree recovered Z. transpicula with Z. triangularis, and comparison with a published nuclear single-copy ortholog tree based on 1962 loci supported a non-mitochondrial placement of Zhengitettix inconsistent with the anomalous mitochondrial position of Z. transpicula. Independent assembly from the original HiFi reads, read-depth inspection, protein-coding gene checks, and nuclear-genome screening for NUMT-like sequences supported the authenticity of the assembled Z. transpicula mitogenome. These results document mito–nuclear and cyto-morphological discordance in Tetrigidae and highlight the need for integrative interpretation of mitochondrial phylogenies in taxonomically complex insect groups. Full article

Leaf architecture critically impacts crop yield. The Auxin Response Factor (ARF) family is a key regulator of leaf development, yet remains uncharacterized in the important legume crop chickpea (Cicer arietinum L.), which bears pinnate compound leaves. Here, we performed a genome-wide identification and analysis of ARF genes in chickpea. We identified 33 CaARF genes and resolved their phylogenetic structure through comparison with six other key dicot species. The analysis revealed a deeply conserved core set of ARF proteins across species, all sharing the N-terminal DNA-binding domain (DBD), with most the C-terminal PB1 domain, connected by a middle region (MR). We also uncovered instances of lineage-specific expansion, e.g., a chickpea-specific ARF clade, which is characterized by the absence of the C-terminal PB1 domain. Expression profiling using public transcriptome data and qRT-PCR revealed distinct spatiotemporal expression patterns for CaARF genes across tissues and during compound leaf development. Detailed in situ hybridization analysis for selected candidates, chosen based on phylogenetic proximity to known leaf-development-related ARFs in other species, localized their transcripts to specific regions within compound leaf primordia. Focusing on CaARF5, the closest ortholog of Arabidopsis MONOPTEROS/ARF5, we confirmed its nuclear localization and dynamic expression during chickpea leaf development. Functional complementation assays demonstrated that CaARF5 could restore developmental defects in the Arabidopsis mp mutant. Our study establishes an evolutionary and molecular framework for the chickpea ARF family, highlighting conserved features and species-specific innovations, and provides essential resources for future research on auxin-mediated leaf development and ARF-targeted legume breeding. Full article

Background: Long noncoding RNAs (lncRNAs) have emerged as critical regulators of hepatic metabolism and disease progression. The hepatocyte nuclear factor 1 alpha antisense 1 (HNF1A-AS1) lncRNA modulates liver-specific transcription factors; however, its physiological role in diet-dependent lipid homeostasis remains poorly defined. Methods: In this study, we investigated the mouse ortholog, Hnf1a opposite strand 1 (Hnf1aos1), using AAV-mediated knockdown in C57BL/6J mice fed either a chow diet (10% kcal from fat) or a high-fat diet (HFD; 60% kcal from fat) for 12 weeks. Metabolic phenotyping included hepatic lipid quantification, histological analysis, serum biochemistry, and quantitative gene expression profiling. Results: Loss of Hnf1aos1 produced distinct, diet-dependent alterations in hepatic lipid handling. Under chow conditions, knockdown mice exhibited selective hepatic cholesterol accumulation (6.10 ± 2.9 mg/g tissue vs. 3.51 ± 1.1 mg/g in controls), accompanied by dysregulation of cholesterol clearance pathways. In contrast, under HFD conditions, knockdown precipitated severe macrovesicular degeneration, with hepatic triglyceride levels approximately doubled relative to HFD-fed controls (51.72 ± 19.8 mg/g vs. 26.34 ± 11.9 mg/g) and a numerically elevated triglyceride-to-cholesterol ratio (TG:TC ≈ 6.1:1; p = 0.0621, trend). Chow/Kd mice gained significantly less weight than chow-fed controls, whereas HFD/Kd mice exhibited weight gain comparable to HFD controls despite severe hepatic steatosis. This paradoxical phenotype suggests impaired metabolic feedback at the post-transcriptional level, in which compensatory upregulation of Hnf1a mRNA is insufficient to suppress lipid-associated genes such as Cd36, despite profound lipid overload; however, HNF1A protein levels were not directly measured in this study. Conclusion: Collectively, these findings identify Hnf1aos1 as a regulator of hepatic lipid homeostasis whose loss produces a phenotype consistent with inappropriate lipid accumulation during nutrient excess, without defining the underlying molecular mechanism. Our results support a role for Hnf1aos1 in shaping hepatic metabolic plasticity and provide insight into lncRNA-associated MASLD phenotypes. Full article

Nuclear Factor Y (NF-Y) transcription factors play pivotal roles in plant adaptation to abiotic stress, yet their genomic landscape and functional mechanisms in cabbage (Brassica oleracea var. capitata L.) remain underexplored. Here, we performed a genome-wide identification of the NF-Ys in cabbage, identifying 53 BoNF-Ys classified into three subfamilies: 20 BoNF-YAs, 22 BoNF-YBs, and 11 BoNF-YCs. Phylogenetic clustering revealed evolutionary conservation with their Arabidopsis orthologs. Domain analysis revealed that all BoNF-YA members contain the CBF_NF-YA domain, while all BoNF-YB and BoNF-YC members possess the CBFD_NFYB_HMF conserved domain. The BoNF-Y genes were named according to their chromosomal locations. Bioinformatic analysis showed that BoNF-Y proteins range in size from 131 to 642 amino acids, with molecular weights of 14.82–73.18 kDa, theoretical pI values of 4.57–9.96, instability indices between 33.02 and 73.48, aliphatic indices of 45.3–86.26, and grand average of hydropathicity (GRAVY) values ranging from −1.139 to −0.367. Promoter cis-element profiling uncovered stress- and hormone-responsive motifs, including abscisic acid-responsive elements (ABREs), TC-rich repeats, and ethylene-responsive elements (EREs). RNA sequencing (RNA-seq) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) conducted under salt stress (256 mM) identified three salt-responsive candidate genes (BoNF-YA14, BoNF-YB9, and BoNF-YC8). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses highlighted significantly expressed genes’ roles in MAPK signaling, proline metabolism, and phytohormone transduction pathways. This study conducted a comprehensive survey of the BoNF-Y gene family in cabbage. It could serve as a theoretical foundation for further functional identification and utilization of BoNF-Y family members and their role in the interaction between cabbage and salt stress. Full article

Nuclear receptor HR97 is considered as a non-insect arthropod–specific receptor, but its roles in decapod reproduction remain poorly understood. Here, we identified and characterized an HR97 ortholog from the swimming crab Portunus trituberculatus (PtHR97) and verified its placement within the NR1L nuclear receptor family by phylogenetic analysis. PtHR97 encodes a canonical nuclear receptor with a conserved DNA-binding domain (DBD) and ligand-binding domain (LBD). Quantitative PCR revealed predominant PtHR97 expression in the ovary and stage-dependent changes during ovarian development. Using an ovarian explant culture system, we found that arachidonic acid (AA) consistently suppressed PtHR97 transcript levels, while methyl farnesoate (MF) and pyriproxyfen (P) had no significant effect, indicating a potential inhibitory role for AA in PtHR97 expression. RNA interference of HR97 caused significant changes in ovarian development, including reduced GSI, smaller oocytes, and uneven eosinophilic granule distribution. Transcriptomic profiling of HR97-silenced ovaries indicated that the major responses involved genes associated with substrate transport/exchange, cell boundary–related signaling and transduction, and disturbed nuclear transcriptional regulation. Short-term in vivo perturbations (HR97 RNAi and AA treatment) further supported these expression changes and revealed that AA- and HR97 RNAi–elicited transcriptional responses only partially overlapped. Taken together, these results suggest that HR97 contributes to ovarian development, potentially through broad transcriptional responses related to transport, signaling, and gene regulation. Although AA may suppress HR97 expression, HR97 does not fully explain AA-mediated regulation of ovarian development. Full article

The Keap1-Nrf2 signaling pathway is a central regulator of transcriptional responses to oxidative stress and is strongly linked to diverse pathologies, particularly cancer. In the cytoplasm, Keap1 (Kelch-like ECH-associated protein 1) promotes proteasomal degradation of Nrf2 (NF-E2–related factor 2). Oxidative stimuli disrupt the Keap1-Nrf2 interaction, facilitating Nrf2 nuclear accumulation and activation of antioxidant and detoxifying genes. Recent evidence suggests that Keap1 family proteins also enter the nucleus, bind chromatin, and regulate transcription, but the underlying mechanisms remain less understood. Here, we show that the Drosophila Keap1 ortholog, dKeap1, accumulates in the nucleus and gradually assembles stable nuclear foci in cells following oxidative treatment. FRAP analyses revealed reduced mobility of dKeap1 within these foci. Both the N-terminal (NTD) and C-terminal (CTD) domains of dKeap1 were required for foci formation. Two intrinsically disordered regions (IDRs) were identified within the CTD, and CTD-YFP fusion proteins readily formed condensates in vitro. Conversely, deletion of the Kelch domain resulted in robust cytoplasmic foci even under basal conditions, and in vitro assays also indicated that the Kelch domain suppresses dKeap1 condensate formation. Together, these findings reveal a novel molecular mechanism for the nuclear function of dKeap1, providing new insight into the broader roles of Keap1 factors in oxidative response, development, and disease. Full article

The Xenopus oocyte has long served as a versatile and powerful model for dissecting the molecular underpinnings of reproductive and developmental processes. Its large size, manipulability, and well-characterized cell cycle states have enabled generations of researchers to illuminate key aspects of oocyte maturation, fertilization, and early embryogenesis. This review provides an integrated overview of the cellular and molecular events that define the Xenopus oocyte’s transition from meiotic arrest to embryonic activation—or alternatively, to programmed demise if fertilization fails. We begin by exploring the architectural and biochemical landscape of the oocyte, including polarity, cytoskeletal organization, and nuclear dynamics. The regulatory networks governing meiotic resumption are then examined, with a focus on MPF (Cdk1/Cyclin B), MAPK cascades, and translational control via CPEB-mediated cytoplasmic polyadenylation. Fertilization is highlighted as a calcium-dependent trigger for oocyte activation. During fertilization in vertebrates, sperm-delivered phospholipase C zeta (PLCζ) is a key activator of Ca²⁺ signaling in mammals. In contrast, amphibian species such as Xenopus lack a PLCZ1 ortholog and instead appear to rely on alternative protease-mediated signaling mechanisms, including the uroplakin III–Src tyrosine kinase pathway and matrix metalloproteinase (MMP)-2 activity, to achieve egg activation. The review also addresses the molecular fate of unfertilized eggs, comparing apoptotic and necrotic mechanisms and their relevance to reproductive health. Finally, we discuss recent innovations in Xenopus-based technologies such as mRNA microinjection, genome editing, and in vitro ovulation systems, which are opening new avenues in developmental biology and translational medicine. By integrating classic findings with emerging frontiers, this review underscores the continued value of the Xenopus model in elucidating the fundamental processes of life’s origin. We conclude with perspectives on unresolved questions and future directions in oocyte and early embryonic research. Full article

Cas9 with specialized temperature adaptations are essential for broadening the application of CRISPR-based genome editing across diverse biological contexts. Although Cas9 orthologs from thermophilic and mesophilic organisms have been characterized for high- and moderate-temperature applications, cold-active variants remain largely unexplored, limiting genome engineering in low-temperature systems such as aquaculture species. Here, we report the functional characterization of Fp2Cas9, a cold-adapted Type II-C nuclease from Flavobacterium psychrophilum. In vitro assays showed that Fp2Cas9 efficiently cleaves double-stranded DNA with a refined PAM requirement of 5′-SNAAAG-3′, and that its engineered sgRNA scaffold (sgRNA-V2) supports programmable DNA targeting. Notably, Fp2Cas9 retains 75% cleavage efficiency at 5 °C, approximately 2.5-fold higher than SpCas9 under the same conditions, but shows a marked reduction in activity at 35 °C. In vivo, a nuclear-localized variant (2NLS-Fp2Cas9) mediated efficient mutagenesis of the zebrafish slc45a2 gene, yielding ~60% indel frequencies and pigmentation-deficient phenotypes in ~43% of injected embryos. Collectively, these findings establish Fp2Cas9 as a cold-adapted Cas9 with reliable activity at low temperatures. This work adds a valuable tool to the CRISPR-Cas9 toolkit and may facilitate genome editing in cold-water organisms and other low-temperature systems. Full article

Epigenetic modifications are long-lasting changes to the genome that influence a cell’s transcriptional potential, thereby altering its function. These modifications can trigger adaptive responses that impact protein expression and various cellular processes, including differentiation and growth. The primary epigenetic mechanisms identified to date include DNA and RNA methylation, histone modifications, and microRNA-mediated regulation of gene expression. The intricate crosstalk among these mechanisms makes epigenetics a compelling field for the development of novel control strategies, particularly through the use of epigenetic drugs targeting arthropod vectors such as ticks. In this study, we identified the Rhipicephalus microplus orthologs of canonical histone-modifying enzymes, along with components of the machinery responsible for m⁵C and ⁶mA-DNA, and m⁶A-RNA methylations. We further characterized their transcriptional profiles and enzymatic activities during embryonic development. To explore the functional consequences of epigenetic regulation in R. microplus, we evaluated the effects of various epigenetic inhibitors on the BME26 tick embryonic cell line. Molecular docking simulations were performed to predict the binding modes of these inhibitors to tick enzymes, followed by in vitro assessment of their effects on cell viability and morphology. Tick cells exposed to these inhibitors presented phenotypic and molecular alterations. Notably, we observed high levels of DNA methylation in the nuclear genome. Importantly, inhibition of DNA methylation using 5′-azacytidine (5′-AZA) was associated with increased activity of the mitochondrial electron transport chain and ATP synthesis but reduced cellular proliferation. Our findings highlight the importance of epigenetic regulation during tick embryogenesis and suggest that targeting these pathways may constitute a novel and promising strategy for tick control. Full article

Chk1, a highly conserved serine/threonine protein kinase, functions as a critical regulator of fungal cell cycle progression, mitotic fidelity, and DNA damage response. In this study, we characterized an orthologous Chk1 (AoChk1) in a ubiquitous nematode-trapping fungus, Arthrobotrys oligospora, through targeted gene knockout coupled with integrated phenotypic, metabolomic, and transcriptomic analyses. This study aims to elucidate the function and potential regulatory networks of AoChk1 in A. oligospora. Deletion of Aochk1 leads to significant reductions in nucleus number, hyphal cell length, conidial production, and trap formation, but an increase in the accumulation of lipid droplets and autophagy. In addition, transcriptomics data indicate that AoChk1 plays an important role in cell cycle and division, nuclear architecture and organelle dynamics, protein homeostasis maintenance, and membrane systems. In addition, the inactivation of the Aochk1 exhibited remarkably reduced metabolite abundance relative to the WT strain. In conclusion, our results identify AoChk1 as an important regulator of asexual development, pathogenicity, and metabolic processes in A. oligospora. Full article

In placental mammals, the co-option of vertebrate orthologous ATP1B4 genes has profoundly altered the properties of the encoded BetaM proteins, which function as bona fide β-subunits of Na,K-ATPases in lower vertebrates. Eutherian BetaM acquired an extended Glu-rich N-terminal domain resulting in the complete loss of its ancestral function and became a skeletal and cardiac muscle-specific component of the inner nuclear membrane. BetaM is expressed at the highest level during perinatal development and is implicated in gene regulation. Here we report the long-term consequences of Atp1b4 ablation on metabolic parameters in adult mice. Male BetaM-deficient (Atp1b4−/Y) mice have remarkably lower body weight and adiposity than their wild-type littermates, despite higher food intake. Indirect calorimetry shows higher energy expenditure (heat production and oxygen consumption) with a greater spontaneous locomotor activity in Atp1b4−/Y males. Their lower respiratory exchange ratio suggests a greater reliance on fat metabolism compared to their wild-type counterparts. Consistently, Atp1b4−/Y KO mice exhibit enhanced β-oxidation in skeletal muscle, along with improved glucose and insulin tolerance. These robust metabolic changes induced by Atp1b4 disruption demonstrate that eutherian BetaM plays an important role in regulating adult mouse metabolism. This demonstrates that bypassing the co-option of Atp1b4 potentially reduces susceptibility to obesity. Thus, Atp1b4 ablation leading to the loss of evolutionarily acquired BetaM functions serves as a model for a potential alternative pathway in mammalian evolution. Full article

Autophagy is an essential eukaryotic catabolic process through which damaged or superfluous cellular components are degraded and recycled via the formation of double-membrane autophagosomes. In plants, autophagy-related genes (ATGs) are primarily expressed in the cytoplasm and are responsible for orchestrating distinct stages of autophagosome biogenesis. Among these, ATG8 proteins, orthologous to the mammalian LC3 family, are conserved ubiquitin-like modifiers that serve as central hubs in selective autophagy regulation. Although ATG8 proteins are localized in both the cytoplasm and nucleus, their functions within the nucleus remain largely undefined. In the present study, the ATG8-interacting motif (AIM) was identified and functionally characterized in the potato ATG8 homolog (StATG8), demonstrating its capacity for selective target recognition. StATG8 was shown to form both homodimeric and heterodimeric complexes with other ATG8 isoforms, implying a broader regulatory potential within the ATG8 family. Notably, StATG8 was found to interact with the Ralstonia solanacearum type III effector PopP2, a nuclear-localized acetyltransferase, suggesting a possible role in effector recognition within the nucleus. In addition, interactions between StATG8 and transcription factors AtWRKY40 and AtWRKY60 were detected in both cytoplasmic autophagosomes and the nuclear compartment. These observations provide novel insights into the noncanonical, nucleus-associated roles of plant ATG8 proteins. The nuclear interactions with pathogen effectors and transcriptional regulators suggest that ATG8 may function beyond autophagic degradation, contributing to the regulation of nuclear signaling and plant immunity. These findings offer a foundational basis for further investigation into the functional diversification of ATG8 in plant cellular compartments. Full article