Nucleic Acids Research最新文献

A central challenge in the quest for precise gene regulation within mammalian cells is the development of regulatory networks that can achieve perfect adaptation-where outputs consistently return to a set baseline post-stimulus. Here, we present such a system that leverages the CRISPR activation (CRISPRa) and anti-CRISPR proteins as two antithetic elements to establish perfect adaptation in mammalian cells and dynamically regulate gene expression. We demonstrate that this system can maintain stable expression levels of target genes in the face of external perturbations, thus providing a robust platform for biological applications. The versatility of our system is further showcased through its integration with endogenous regulatory mechanisms in T cells, such as the NF-κB-mediated immune response, and its ability to program apoptosis responses for precise spatial and temporal control of cellular growth and death. This study not only advances our understanding of gene regulation in mammalian cells but also opens new avenues for therapeutic intervention, particularly in diseases characterized by dysregulated gene expression.

The determination of the three-dimensional structure of large RNA macromolecules in solution is a challenging task that often requires the use of several experimental and computational techniques. Small-angle X-ray scattering can provide insight into some geometrical properties of the probed molecule, but this data must be properly interpreted in order to generate a three-dimensional model. Here, we propose a multiscale pipeline which introduces SAXS data into modelling the global shape of RNA in solution, which can be hierarchically refined until reaching atomistic precision in explicit solvent. The low-resolution helix model (Ernwin) deals with the exploration of the huge conformational space making use of the SAXS data, while a nucleotide-level model (SPQR) removes clashes and disentangles the proposed structures, leading the structure to an all-atom representation in explicit water. We apply the procedure on four different known pdb structures up to 159 nucleotides with promising results. Additionally, we predict an all-atom structure for the Plasmodium falceparum signal recognition particle ALU RNA based on SAXS data deposited in the SASBDB, which has an alternate conformation and better fit to the SAXS data than the previously published structure based on the same data but other modelling methods.

Monoubiquitination of histones H2B-K120 (H2BK120ub) and H2A-K119 (H2AK119ub) play opposing roles in regulating transcription and chromatin compaction. H2BK120ub is a hallmark of actively transcribed euchromatin, while H2AK119ub is highly enriched in transcriptionally repressed heterochromatin. Whereas H2BK120ub is known to stimulate the binding or activity of various chromatin-modifying enzymes, this post-translational modification (PTM) also interferes with the binding of several proteins to the nucleosome H2A/H2B acidic patch via an unknown mechanism. Here, we report cryoEM structures of an H2BK120ub nucleosome showing that ubiquitin adopts discrete positions that occlude the acidic patch. Molecular dynamics simulations show that ubiquitin remains stably positioned over this nucleosome region. By contrast, our cryoEM structures of H2AK119ub nucleosomes show ubiquitin adopting discrete positions that minimally occlude the acidic patch. Consistent with these observations, H2BK120ub, but not H2AK119ub, abrogates nucleosome interactions with acidic patch-binding proteins RCC1 and LANA, and single-domain antibodies specific to this region. Our results suggest a mechanism by which H2BK120ub serves as a gatekeeper to the acidic patch and point to distinct roles for histone H2AK119 and H2BK120 ubiquitination in regulating protein binding to nucleosomes.

Gene clusters are genomic loci that contain multiple genes that are functionally and genetically linked. Gene clusters collectively encode diverse functions, including small molecule biosynthesis, nutrient assimilation, metabolite degradation, and production of proteins essential for growth and development. Identifying gene clusters is a powerful tool for small molecule discovery and provides insight into the ecology and evolution of organisms. Current detection algorithms focus on canonical 'core' biosynthetic functions many gene clusters encode, while overlooking uncommon or unknown cluster classes. These overlooked clusters are a potential source of novel natural products and comprise an untold portion of overall gene cluster repertoires. Unbiased, function-agnostic detection algorithms therefore provide an opportunity to reveal novel classes of gene clusters and more precisely define genome organization. We present CLOCI (Co-occurrence Locus and Orthologous Cluster Identifier), an algorithm that identifies gene clusters using multiple proxies of selection for coordinated gene evolution. Our approach generalizes gene cluster detection and gene cluster family circumscription, improves detection of multiple known functional classes, and unveils non-canonical gene clusters. CLOCI is suitable for genome-enabled small molecule mining, and presents an easily tunable approach for delineating gene cluster families and homologous loci.

Lipid nanoparticles (LNPs) have been demonstrated to hold great promise for the clinical advancement of RNA therapeutics. Continued exploration of LNPs for application in new disease areas requires identification and optimization of leads in a high throughput way. Currently available high throughput in vivo screening platforms are well suited to screen for cellular uptake but less so for functional cargo delivery. We report on a platform which measures functional delivery of LNPs using unique peptide 'barcodes'. We describe the design and selection of the peptide barcodes and the evaluation of these for the screening of LNPs. We show that proteomic analysis of peptide barcodes correlates with quantification and efficacy of barcoded reporter proteins both in vitro and in vivo and, that the ranking of selected LNPs using peptide barcodes in a pool correlates with ranking using alternative methods in groups of animals treated with individual LNPs. We show that this system is sensitive, selective, and capable of reducing the size of an in vivo study by screening up to 10 unique formulations in a single pool, thus accelerating the discovery of new technologies for mRNA delivery.

DNA-protein crosslinks (DPCs) challenge faithful DNA replication and smooth passage of genomic information. Our study unveils the cullin E3 ubiquitin ligase Rtt101 as a DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types including topoisomerase 1 crosslinks, in the same pathway as the ubiquitin-dependent aspartic protease Ddi1. Using an in vivo inducible Top1-mimicking DPC system, we reveal the significant impact of Rtt101 ubiquitination on DPC removal across different cell cycle phases. High-throughput methods coupled with next-generation sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a main contributor to DPC repair throughout the yeast cell cycle.

DNA damage has been implicated in the stimulation of the type 1 interferon (T1IFN) response. Here, we show that downregulation of the DNA repair protein, polynucleotide kinase/phosphatase (PNKP), in a variety of cell lines causes robust phosphorylation of STAT1, upregulation of interferon-stimulated genes and persistent accumulation of cytosolic DNA, all of which are indicators for the activation of the T1IFN response. Furthermore, this did not require damage induction by ionizing radiation. Instead, our data revealed that production of reactive oxygen species (ROS) synergises with PNKP loss to potentiate the T1IFN response, and that loss of PNKP significantly compromises mitochondrial DNA (mtDNA) integrity. Depletion of mtDNA or treatment of PNKP-depleted cells with ROS scavengers abrogated the T1IFN response, implicating mtDNA as a significant source of the cytosolic DNA required to potentiate the T1IFN response. The STING signalling pathway is responsible for the observed increase in the pro-inflammatory gene signature in PNKP-depleted cells. While the response was dependent on ZBP1, cGAS only contributed to the response in some cell lines. Our data have implications for cancer therapy, since PNKP inhibitors would have the potential to stimulate the immune response, and also to the neurological disorders associated with PNKP mutation.

The endonuclease activity of Pms1 directs mismatch repair by generating a nick in the newly replicated DNA strand. Inactivating Pms2, the human homologue of yeast Pms1, increases the chances of colorectal and uterine cancers. Here we use whole genome sequencing to show that loss of this endonuclease activity, via the pms1-DE variant, results in strong mutator effects throughout the Saccharomyces cerevisiae genome. Mutation rates are strongly increased for mutations resulting from all types of single-base substitutions and for a wide variety of single- and multi-base indel mutations. Rates for these events are further increased in strains combining pms1-DE with mutator variants of each of the three major leading and lagging strand replicases. In all cases, mutation rates, spectra, biases, and context preferences are statistically indistinguishable from strains with equivalent polymerases but lacking initial mismatch recognition due to deletion of MSH2. This implies that, across the nuclear genome, strand discrimination via the Pms1 endonuclease is as important for MMR as is initial mismatch recognition by Msh2 heterodimers.

Recently, we introduced a chromatin immunoprecipitation (ChIP) technique utilizing the human DNA Fragmentation Factor (DFF) to digest the DNA prior to immunoprecipitation (DFF-ChIP) that provides the precise location of transcription complexes and their interactions with neighboring nucleosomes. Here we expand the technique to new targets and provide useful information concerning purification of DFF, digestion conditions, and the impact of crosslinking. DFF-ChIP analysis was performed individually for subunits of Mediator, DSIF, and NELF that that do not interact with DNA directly, but rather interact with RNA polymerase II (Pol II). We found that Mediator was associated almost exclusively with preinitiation complexes (PICs). DSIF and NELF were associated with engaged Pol II and, in addition, potential intermediates between PICs and early initiation complexes. DFF-ChIP was then used to analyze the occupancy of a tight binding transcription factor, CTCF, and a much weaker binding factor, glucocorticoid receptor (GR), with and without crosslinking. These results were compared to those from standard ChIP-Seq that employs sonication and to CUT&RUN which utilizes MNase to fragment the genomic DNA. Our findings indicate that DFF-ChIP reveals details of occupancy that are not available using other methods including information revealing pertinent protein:protein interactions.

Long terminal repeat (LTR)-retrotransposons are significant contributors to the evolution and diversity of eukaryotic genomes. Their RNA genomes (gRNA) serve as a template for protein synthesis and reverse transcription to a DNA copy, which can integrate into the host genome. Here, we used the SHAPE-MaP strategy to explore Ty3 retrotransposon gRNA structure in yeast and under cell-free conditions. Our study reveals the structural dynamics of Ty3 gRNA and the well-folded core, formed independently of the cellular environment. Based on the detailed map of Ty3 gRNA structure, we characterized the structural context of cis-acting sequences involved in reverse transcription and frameshifting. We also identified a novel functional sequence as a potential initiator for Ty3 gRNA dimerization. Our data indicate that the dimer is maintained by direct interaction between short palindromic sequences at the 5' ends of the two Ty3 gRNAs, resembling the model characteristic for other retroelements like HIV-1 and Ty1. This work points out a range of cell-dependent and -independent Ty3 gRNA structural changes that provide a solid background for studies on RNA structure-function relationships important for retroelement biology.