Nucleic Acids Research最新文献

In human cells, the Nuclear EXosome Targeting (NEXT) and Poly(A) tail eXosome Targeting (PAXT) adaptors direct the nuclear exosome to degrade prematurely terminated RNA Polymerase II (Pol II) transcripts, ensuring nuclear RNA quality control. How these adaptors interact with transcription termination machineries remains largely unclear. Here, we leveraged in silico structure predictions of protein complexes to identify and model previously unreported interactions of NEXT- and PAXT-associated components with two transcription termination and processing machineries, the Integrator and Cleavage and Polyadenylation (CPA) complexes. Our computational models were validated through complementary in vitro biochemical approaches and single-particle cryo-EM analyses. We show that the ZC3H18 protein uses two different domains to directly recognize the INTS9/11 endonuclease module of Integrator and the mammalian Polyadenylation Specificity Factor (mPSF), a core CPA component. In turn, ZC3H18 can directly bind the scaffolding subunits of NEXT and PAXT via mutually exclusive interactions. Furthermore, we provide evidence that accessory PAXT components can be directly integrated with the mPSF core, establishing configurations that are mutually exclusive with those of canonical CPA subunits. These findings reveal a versatile interaction network capable of forming alternative structural frameworks linking transcription termination with nuclear RNA quality control.

DNMT1 is a methyltransferase that restores 5-methylcytidine marks on newly replicated DNA and is required for maintaining epigenetic inheritance. Using Halo-tagged DNMT1 and highly inclined thin illumination (HiLo) microscopy, we show that DNMT1 mobility in living human cells changes under a variety of conditions. DNMT1 molecules become increasingly bound to chromatin in the S phase of the cell cycle, but surprisingly only ∼ 12% chromatin-bound DNMT1 is sufficient to maintain DNA methylation. Upon treatment with small molecule inhibitors, GSK-3484862 (GSK), 5-azacytidine (5-azaC) and decitabine (5-aza-deoxyC), in vivo DNMT1 dynamics are greatly altered. Unexpectedly, treatment of cells with GSK, a non-covalent inhibitor, causes binding of DNMT1 to chromatin similar to that observed upon treatment with 5-azaC and decitabine, covalent inhibitors. 5-azaC inhibition of DNMT1 dynamics occurs during the S phase of the cell cycle. Unexpectedly, mutations in the disordered, Asp- and Glu-rich N-terminal region of DNMT1 dramatically decrease its mobility and increase chromatin binding. Collectively, our work using live cell single molecule imaging quantifies the molecular dynamics of DNMT1 and how this relates to its function under physiological conditions and upon drug treatment. Understanding the dynamics of DNMT1 in vivo provides a framework for developing better therapeutics that target DNMT1.

HNRNPU is an RNA-binding protein with diverse roles in transcriptional and post-transcriptional regulation. Pathogenic genetic variants of HNRNPU cause a severe neurodevelopmental disorder (NDD), but the underlying molecular mechanisms are unclear. Here, we comprehensively investigate the HNRNPU molecular interactome by integrating protein-protein interaction (PPI) mapping, RNA target identification, and genome-wide DNA methylation profiling in human neuroepithelial stem cells and differentiating neural cells. We identified extensive HNRNPU-centered networks, including an association with the mammalian SWI/SNF chromatin-remodeling complex, and uncovered a previously unrecognized role in translation. We present evidence that HNRNPU associates with messenger RNAs (mRNAs) encoding proteins important for neuronal development, including several linked to NDDs. Silencing HNRNPU reprogrammed methylation dynamics at regulatory regions, particularly at active and bivalent promoters of neurodevelopmental transcription factors. Integrative analysis across PPI, RNA, and methylome datasets identified 19 converging genes at all three molecular levels, including NDD genes within the SWI/SNF complex, SMARCA4 and SMARCC2, and RNA-processing machinery such as SYNCRIP. Together, these data showcase HNRNPU as a central coordinator of RNA metabolism and epigenetic remodeling during neural differentiation, linking RNA-binding, chromatin organization, and DNA methylation to the pathogenesis of HNRNPU-related NDDs.

Despite the many advances in single cell genomics, detecting structural rearrangements in single cells, particularly error-free sister-chromatid exchanges, remains challenging. Here we describe sci-L3-Strand-seq, a combinatorial indexing method with linear amplification for DNA template strand sequencing that cost-effectively scales to millions of single cells, as a platform for mapping mitotic crossover (CO) and resulting genome instability events. We provide a computational framework to fully leverage the throughput, as well as the relatively sparse but multifaceted genotype information within each cell that includes strandedness, digital counting of copy numbers, and haplotype-aware chromosome segmentation, to systematically distinguish seven possible types of mitotic CO outcomes. We showcase the power of sci-L3-Strand-seq by quantifying the rates of error-free and mutational COs in thousands of cells, enabling us to explore enrichment patterns of genomic and epigenomic features. The throughput of sci-L3-Strand-seq also gave us the ability to measure subtle phenotypes, opening the door for future large mutational screens. Furthermore, mapping clonal lineages provided insights into the temporal order of certain genome instability events, showcasing the potential to dissect cancer evolution. Altogether, we show the wide applicability of sci-L3-Strand-seq to the study of DNA repair and structural variations.

Cyanobacteria are one of the oldest and most abundant groups of prokaryotes and are crucial for research in climate, ecology, medicine, and agriculture. Despite intensive efforts in metabolic engineering of cyanobacteria, the mechanisms of gene regulation, particularly through regulatory RNA structures, are often ignored. We computationally searched 202 cyanobacterial genomes for putative conserved RNA structures (CRSs) in the upstream and downstream intergenic regions of 931 orthologous gene groups with the comparative genomics tool CMfinder. The predicted structures were scored according to their local phylogeny and filtered for a maximal false discovery rate of 10%. The screen identified 402 CRSs that match known RNA families (Rfam and Rho-independent bacterial terminators) and 409 novel CRSs. The structures are not limited to either low or high nucleotide conservation, and about half have a high level of significant covariation. The majority of novel CRSs are supported by transcription in at least one species in public RNA-seq data. The regulatory associations of CRSs are discussed in different metabolic pathways, such as photosynthesis, nitrogen fixation, and CO$_2$ metabolism. This resource will support future research on the regulatory mechanisms of RNA in cyanobacteria.

Integrases serve as powerful biotechnology tools that catalyze recombination at specific DNA sequences (att sites) and facilitate chromosomal integration of gene cargos transferred into cells. Given that genomes often lack the attB integration sites recognized by frequently utilized integrases, integrase technology has largely been restricted to genetic engineering of model organisms into which attB sites can be synthetically introduced. To enable single-step site-specific integrase-mediated genome editing in a broad spectrum of prokaryotes, we have devised the Integrase-On-Demand (IOD) method. IOD systematically identifies integrases, within bacteria and archaea, that can integrate into available attB sites in any target prokaryote. Computational results show that diverse bacteria generally have multiple potentially useable native attB sites for novel integrases. We confirmed the functionality of predicted integrase and attB pairs for mediating site-specific integration of heterologous DNA into the genomes of Pseudomonas putida S12 and KT2440 and Synechococcus elongatus UTEX 2973, measuring efficiency of integration using nonreplicating vectors. By eliminating the requirement to introduce non-native attB sites into the target genome, IOD may, when suitable transformation methods exist, allow facile genome integration of large constructs in nonmodel and possibly nonculturable bacteria.

The mammalian SWI/SNF family of chromatin remodelers comprises BRG1/BRM-associated factor (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF) complexes, which slide and disassemble nucleosomes to regulate gene expression and chromatin structure dependent on ATP hydrolysis energy. While the chromatin engagement mechanisms of cBAF and PBAF have been structurally resolved, the molecular architecture governing ncBAF interaction with chromatin remains elusive. In this study, by integrating cryo-electron microscopy, biochemical assays, and cross-linking mass spectrometry, we resolved the conformational transition of ncBAF-nucleosome complexes from nucleotide-free to nucleotide-bound states. Our analyses establish BCL7 proteins as dynamic molecular tethers connecting the ARP module to the nucleosomal acidic patch and demonstrate that BCL7B promotes ncBAF-mediated nucleosome remodeling, with BRG1-catalyzed ATP hydrolysis triggering conformational changes that modulate BCL7-mediated histone association. Structurally and biochemically, we further demonstrate that β-actin within the BCL7-containing ARP module retains ATP hydrolysis activity, rendering its exposed pointed end structurally compatible with incorporation into the barbed end of nuclear actin filaments, which provides a potential molecular basis for coordinating nuclear actin networks with chromatin remodeling. Collectively, our findings unravel a dynamic role of BCL7 in regulating ncBAF-mediated chromatin remodeling and establish a distinct chromatin engagement mode of ncBAF from that of cBAF/PBAF.

Emerging models of nuclear organization suggest that chromatin forms functionally distinct microenvironments through phase separation. As chromatin architecture is organized at the level of the nucleosome and regulated by histone post-translational modifications, we investigated how these known regulatory mechanisms influence nucleosome phase behavior. By systematically altering charge distribution within the H3 tail, we found that the terminal and central regions modulate the phase boundary and tune nucleosome condensate viscosity differentially, as revealed by microscopy-based assays, microrheology, and simulations. Nuclear magnetic resonance relaxation experiments revealed that H3 tails remain dynamically mobile within condensates, and their mobility correlates with condensate viscosity. These results demonstrate that the number, identity, and spatial arrangement of basic residues in the H3 tail critically regulate nucleosome phase separation. Our findings support a model in which nucleosomes, through their intrinsic properties and modifications, actively shape the local chromatin microenvironment-providing new insight into the histone language in chromatin condensates.

Non-genetic variability in gene expression is an inevitable consequence of the stochastic nature of processes driving transcription and translation. While previous studies demonstrated that gene expression noise is negatively correlated with gene body methylation, the function of this correlation remains poorly understood in multicellular systems. Here, we provide a first functional link between gene body methylation and transcription noise in plants. We investigated a mutant with partial loss of CG methylation (met1-1) and 10 epigenetic recombinant inbred lines (epiRILs) generated by a cross between Col-0 and met1-3 plants, and observed an increase in gene expression noise, but this was not the case in met1-3 with complete loss of CG methylation. Loss of CG methylation in met1-3 could be compensated by a low but significant gain of non-CG methylation that buffers the noise in gene expression. Overall, our results show that gene body methylation has a functional role in reducing variability in transcription in a large subset of housekeeping genes, which require precise expression patterns to meet metabolic requirements. Genes lacking this noise-buffering effect are mainly enriched in stress response, where variability in gene expression can be seen as highly beneficial.

While DNA nanotechnology holds transformative potential across biomedical and information storage applications, current technologies face critical limitations in synthesizing long single-stranded DNA (ssDNA) with high purity and homogeneity. To address these challenges, we developed Ouroborosyn-ssDNA, a nicking enzymatic assisted replication (NEAR) platform that synergizes enzymatic engineering with computational optimization. By integrating phi29 DNA polymerase and Nb.BbvCI nickase in formate-based buffers, we achieved extended ssDNA synthesis up to 15 000 nt while preserving sequence fidelity, resulting in a 4.73-fold yield enhancement compared to commercial buffers. Notably, machine learning-guided parameter optimization identified magnesium ion dynamics and thermal modulation as pivotal determinants of enzymatic efficiency. Furthermore, solid-phase synthesis using thiol-gold immobilized templates demonstrated 86.38% purification recovery via automated magnetic bead systems, enabling scalable production. To validate functional utility, we engineered six-helix bundle DNA origami-CRISPR complexes that achieved nucleolin-targeted genome editing in cervical cancer cells, coupling GFP-based diagnostics with therapeutic E7 oncogene disruption. These advancements directly overcome key limitations in enzymatic stochasticity and product heterogeneity through buffer engineering and computational optimization, establishing a scalable pathway for applications in precision nanomedicine, synthetic biology, and molecular data storage. This integrated strategy advances DNA nanotechnology from proof-of-concept studies toward standardized biomanufacturing of sequence-defined macromolecular architectures.