Nucleic Acids Research最新文献

SET, the nuclear proto-oncogene, is primarily expressed as SETα in embryonic stem cells (ESCs). Upon pluripotency exit, a transcriptional switch driven by alternative promoters causes SETβ to largely replace SETα expression. Functional distinctions between the two isoforms have been difficult to ascertain, partly due to the redundancy between SETα and SETβ in their protein structure and activity. In this study, we use ESCs with inducible SET isoform-specific expression to investigate the differences between both SET isoforms. Time-course RNA-sequencing analyses in SET-knockout backgrounds as well as isoform-specific chromatin immunoprecipitation followed by sequencing experiments reveal regulatory functions for SETα and SETβ. Despite sharing many binding sites and binding partners, SETα has unique regulatory functions on its target genes, while SETβ downregulates FGF4. As KLF5 specifically regulates SETα, this implicates SET isoform switching at the KLF5/FGF signalling axis during primitive endoderm specification. Together, we propose a model of how distinct roles of SETα and SETβ may regulate cell identity in the early blastocyst.

RNA is subject to many modifications, from small chemical changes like methylation to conjugation of biomolecules such as glycans. As well as endogenously written modifications, RNA is also exposed to damage induced by its environment. Certain clinical compounds are known to covalently modify RNA with a growing appreciation of how these impact clinical efficacy. To understand the regulation of these modifications, we need a reliable, sensitive, and rapid methodology for their quantification. Thus, we developed Aqueous Identification of RNA Elements (AquIRE) and applied it to the analysis of drug-induced RNA damage by 5FU, oxaliplatin, and temozolomide in clinically relevant cell models. We demonstrate that RNA damage is widespread and follows previously unappreciated temporal dynamics. AquIRE also provides a highly sensitive method to detect RNAs modified by glycans. We leverage this to expand the horizons of the glycoRNA world across the kingdoms of life as well as identifying cell-free glycoRNAs in multiple species. We demonstrate that glycoRNA expression is dynamic during embryo development, modulated during senescence, and elevated by RNA-damaging agents. Finally, we use RNA digestion to demonstrate that cell surface or cell-free RNA promotes the cytotoxicity of RNA-damaging chemotherapy. Together, the AquIRE platform provides an intrinsically flexible method to study diverse RNA modifications from any sample.

Gene regulatory mechanisms control cell differentiation and homeostasis but are often undetectable, particularly at the single-cell level. We introduce bayesReact, which quantifies regulatory activities from bulk or single-cell omics data. It is based on an unsupervised generative model, exploiting the fact that each regulator typically targets many genes sharing a sequence motif. Using mRNA expression data, we illustrate and evaluate bayesReact on microRNAs (miRNAs). It outperforms existing methods on sparse bulk data and improves activity inference on single-cell data. Inferred miRNA activities correlate with miRNA expression across pan-cancer TCGA and healthy GTEx tissue samples. The activities capture cancer-type-specific miRNA patterns, e.g., for miR-122-5p and miR-124-3p, which also correlate more strongly with their target genes than their measured expression. This includes a strong negative correlation between miR-124-3p and the anti-neuronal REST transcription factor in nervous system cancers. Analyzing single-cell data, bayesReact detects prominent miRNAs during murine stem cell differentiation, including miR-298-5p, miR-92-2-5p, and the Sfmbt2 cluster (miR-297-669). Furthermore, spatio-temporal inference shows increasing miR-124-3p activity in differentiating neurons during embryonic spinal cord development in mice. bayesReact enables large-scale hypothesis-generating screens for novel regulatory factors and the discovery of condition-specific activities. It is implemented as a user-friendly R package (https://github.com/JakobSkouPedersenLab/bayesReact).

N 6-methyladenosine (m6A) and N6, 2'-O-dimethyladenosine (m6Am) are two RNA modifications that play essential roles in diverse RNA metabolic processes and functions. Despite their importance, the dynamic landscapes and regulatory patterns of m6A and m6Am during the oocyte-to-embryo transition (OET) in humans and mice remain elusive. Here, we developed a highly sensitive method, MeLACE-seq, to profile the m6A and m6Am landscapes across human and mouse oocytes to pre-implantation embryos. We reveal that the zygotic genome activation (ZGA) stage serves as a regulatory node where both the m6A and m6Am methylomes undergo dramatic, species-specific changes. Moreover, transcripts marked by m6A and m6Am are generally expressed and translated at higher levels than unmarked transcripts. Additionally, we discovered that m6A modifications are extensively deposited on human retrotransposon RNAs. These m6A marks exhibit a functional shift, showing a positive correlation with elevated retrotransposon RNA levels in pre-ZGA embryos, but a negative correlation with their expression around the ZGA stage. Together, these findings reveal conserved and species-specific regulatory patterns of the epitranscriptome during human and mouse OETs, providing new insights into the roles of RNA modifications in embryogenesis.

Nonsense mutations, resulting from a premature termination codon (PTC), make up ∼11% of all genetic lesions causing disease, affecting millions of people worldwide. Nonsense suppressor anticodon-edited transfer RNAs (ACE-tRNAs) have emerged as a therapeutic modality for the rescue of PTCs. Delivery of ACE-tRNAs in vivo has been achieved by adeno-associated viral vector and RNA-lipid nanoparticle; however, due to drawbacks associated with these approaches, DNA delivery remains an attractive approach. DNA-based approaches afford ease of manufacturing at a relatively low cost and exhibit improved therapeutic durability and safety as compared to viral vector- or RNA-based approaches. Due to the small size of human tRNA genes employed as ACE-tRNAs, in principle, DNA vectors <200 base pairs (bp) in size (minivectors) could be utilized for delivery of actively transcribed ACE-tRNAs. Here, we demonstrate that linear DNA ACE-tRNA vectors as small as 200 bp effectively suppress several nonsense mutations in CFTR and REP1, and that ACE-tRNA minivectors, when tested in cell or ex vivo models, display significantly improved bioavailability, reduced innate immune burden, and superior biostability as compared to conventional plasmid DNA vectors.

CRISPR-Cas9 knock-in efficiency is often limited by geometric misalignment between donor DNA and the endogenous strand-invasion path. In Aspergillus nidulans, we found that integration drops sharply when the insertion site is offset from the invasion entry point, producing premature annealing or unsupported 3' ends that stall DNA synthesis. Chromatin immunoprecipitation-based profiling shows directional loading of the RAD51 homolog UvsC around Cas9-induced double-strand breaks, thereby defining the spatial origin of strand invasion. Guided by this insight, we introduce a dual-single-guide RNA design that places two cuts flanking the insertion site to create a geometry-matched strand-invasion window. This alignment consistently and markedly increases homology-directed-repair-mediated integration across insert sizes and editing tasks-including C-terminal tagging, bidirectional promoter rewiring, and long-distance dual-site mutagenesis-and generalizes across multiple fungal species. We propose a structural-docking model in which pairing fidelity between the resected chromosomal strand and donor homology arms governs knock-in outcomes, providing a practical design principle for efficient and precise genome engineering at structurally constrained loci.

DNA is most often found in its canonical B-form double-helical structure, but can also adopt alternative conformations, known as non-B DNA structures. Numerous non-B structures have been characterized, including G-quadruplexes, i-motifs, Z-DNA, hairpins, cruciforms, slipped structures, R-loops, and H-DNA. Non-B DNA motifs are enriched in functional regions, including near transcription start and end sites, topologically associated domains, and replication origins, suggesting their importance in gene regulation, genome organization, and replication. However, these structures are intrinsically prone to error-generating processing, leading to genomic instability and hence have been implicated in the development of human diseases. Here, we discuss recent advances in understanding the biological roles of non-B DNA structures and their contribution to genomic instability in somatic and germline contexts. We highlight how they promote replication stress, transcription stalling, and DNA breaks, resulting in the formation of mutational hotspots. Emerging technologies have enabled the detailed mapping of previously challenging repetitive regions that harbor potential non-B DNA-forming sequences, and are poised to unravel additional contributions in human disease and evolution. Furthermore, we explore the dual role of non-B DNA as a driver of genetic variation that facilitates evolutionary adaptation and as a source of mutations that contribute to tissue dysfunction and aging.

Nucleotide salvage is crucial for maintaining DNA replication when de novo nucleotide synthesis is limited, but this metabolic flexibility poses potential threats to genome stability. Salvage kinases phosphorylate nucleosides broadly, allowing for oxidized and alkylated 2'-deoxynucleosides as well as posttranscriptionally modified ribonucleosides to enter the 2'-deoxynucleoside triphosphate (dNTP) pool. The ensuing contamination of the dNTP pool and the subsequent incorporation of modified nucleotides into genomic DNA promote mutagenesis, induce replication stress, elicit double-strand breaks, and disrupt epigenetic signaling. Although only a small subset of modified nucleosides have been assessed for salvage and genomic incorporation, the scope of salvageable substrates is probably much wider, with significant implications in mutational burden, chromatin instability, and epigenetic regulation. This overlooked aspect of genome instability is especially relevant in biological contexts of high salvage activity or elevated nucleoside damage, including chronic inflammation, cancer, aging, and dietary/microbiome exposures. Emerging evidence links salvage metabolism to tumor progression, where incorporation of salvage-derived nucleotides may contribute to unexplainable mutational signatures detected in cancers, such as gastrointestinal cancer. Recognizing salvage as a hidden source of mutagenesis reshapes our understanding of genome instability and provides potential opportunities for disease prevention, diagnosis, and therapeutic intervention.

Z-DNA is known to be a left-handed alternative form of DNA and has important biological roles in cancer and other genetic diseases. In a recent study, we discovered CBL0137, a curaxin ligand, to enhance cancer immunotherapy by inducing Z-DNA formation and activating the Z-DNA-binding protein ZBP1. However, the structural information on binding complexes between Z-DNA and CBL0137 ligand has not reported to date. Here we present the first high-resolution structure of the complex between a Z-DNA and a curaxin ligand CBL0137. This compound is observed to interact with the Z-DNA through π-stacking and zig-zag localization. Furthermore, we directly observe the complex in living human cells using in-cell 19F NMR for the first time. This structural information provides a platform for the design of topology-specific Z-DNA-targeting compounds and is valuable for the development of new potent anticancer drugs.

Heterochromatin is characterized by an inaccessibility to the transcriptional machinery and is associated with the histone mark H3K9me3. However, studying the functional consequences of heterochromatin loss in human cells has been challenging. Here, we used CRISPRi-mediated silencing of the histone methyltransferase SETDB1 to remove H3K9me3 heterochromatin in human neural progenitor cells. Despite a major loss of H3K9me3 peaks resulting in genome-wide reorganization of heterochromatin domains, silencing of SETDB1 had a limited effect on cell viability. Cells remained proliferative and expressed appropriate marker genes. We found that a key event following the loss of SETDB1-mediated H3K9me3 was the expression of evolutionarily young L1 retrotransposons. Derepression of L1s was associated with a loss of CpG DNA methylation at their promoters, suggesting that deposition of H3K9me3 at the L1 promoter is required to maintain DNA methylation. In conclusion, these results demonstrate that loss of H3K9me3 in human neural somatic cells transcriptionally activates evolutionary young L1 retrotransposons.