Nucleic Acids Research最新文献

Until the late 2000s, lactococci substantially contributed to the discovery of various plasmid-borne phage defence systems, rendering these bacteria an excellent antiphage discovery resource. Recently, there has been a resurgence of interest in identifying novel antiphage systems in lactic acid bacteria owing to recent reports of so-called 'defence islands' in diverse bacterial genera. Here, 321 plasmid sequences from 53 lactococcal strains were scrutinized for the presence of antiphage systems. Systematic evaluation of 198 candidates facilitated the discovery of seven not previously described antiphage systems, as well as five systems, of which homologues had been described in other bacteria. All described systems confer resistance against the most prevalent lactococcal phages, and act post phage DNA injection, while all except one behave like abortive infection systems. Structure and domain predictions provided insights into their mechanism of action and allow grouping of several genetically distinct systems. Although rare within our plasmid collection, homologues of the seven novel systems appear to be widespread among bacteria. This study highlights plasmids as a rich repository of as yet undiscovered antiphage systems.

Heterochromatin is a key feature of eukaryotic genomes and is crucial for maintaining genomic stability. In fission yeast, heterochromatin nucleation is mainly mediated by DNA-binding proteins or the RNA interference (RNAi) pathway. In the filamentous fungus Neurospora crassa, however, the mechanism that causes the initiation of heterochromatin at the relics of repeat-induced point mutation is unknown and independent of the classical RNAi pathway. Here, we show that casein kinase II (CKII) and its kinase activity are required for heterochromatin formation at the well-defined 5-kb heterochromatin of the 5H-cat-3 region and transcriptional repression of its adjacent cat-3 gene. Similarly, mutation of the histone H3 phosphorylation site T11 also impairs heterochromatin formation at the same locus. The catalytic subunit CKA colocalizes with H3T11 phosphorylation (H3pT11) within the 5H-cat-3 domain and the deletion of cka results in a significant decrease in H3T11 phosphorylation. Furthermore, the loss of kinase activity of CKII results in a significant reduction of H3pT11, H3K9me3 (histone H3 lysine 9 trimethylation) and DNA methylation levels, suggesting that CKII regulates heterochromatin formation by promoting H3T11 phosphorylation. Together, our results establish that histone H3 phosphorylation by CKII is a critical event required for heterochromatin formation.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

Trichoderma reesei is an economically important enzyme producer with several unique meiotic features. spo11, the initiator of meiotic double-strand breaks (DSBs) in most sexual eukaryotes, is dispensable for T. reesei meiosis. T. reesei lacks the meiosis-specific recombinase Dmc1. Rad51 and Sae2, the activator of the Mre11 endonuclease complex, promote DSB repair and chromosome synapsis in wild-type and spo11Δ meiosis. DNA methyltransferases (DNMTs) perform multiple tasks in meiosis. Three DNMT genes (rid1, dim2 and dimX) differentially regulate genome-wide cytosine methylation and C:G-to-T:A hypermutations in different chromosomal regions. We have identified two types of DSBs: type I DSBs require spo11 or rid1 for initiation, whereas type II DSBs do not rely on spo11 and rid1 for initiation. rid1 (but not dim2) is essential for Rad51-mediated DSB repair and normal meiosis. rid1 and rad51 exhibit a locus heterogeneity (LH) relationship, in which LH-associated proteins often regulate interconnectivity in protein interaction networks. This LH relationship can be suppressed by deleting dim2 in a haploid rid1Δ (but not rad51Δ) parental strain, indicating that dim2 and rid1 share a redundant function that acts earlier than rad51 during early meiosis. In conclusion, our studies provide the first evidence of the involvement of DNMTs during meiotic initiation and recombination.

Mitochondrial transcripts in Trypanosoma brucei require extensive uridine insertion/deletion RNA editing to generate translatable open reading frames. The RNA editing substrate binding complex (RESC) serves as the scaffold that coordinates the protein-protein and protein-RNA interactions during editing. RESC broadly contains two modules termed the guide RNA binding complex (GRBC) and the RNA editing mediator complex (REMC), as well as organizer proteins. How the protein and RNA components of RESC dynamically interact to facilitate editing is not well understood. Here, we examine the roles of organizer proteins, RESC8 and RESC14, in facilitating RESC dynamics. High-throughput sequencing of editing intermediates reveals an overlapping RESC8 and RESC14 function during editing progression across multiple transcripts. Blue native PAGE analysis demonstrates that RESC14 is essential for incorporation of RESC8 into a large RNA-containing complex, while RESC8 is important in recruiting a smaller ribonucleoprotein complex (RNP) to this large complex. Proximity labeling shows that RESC14 is important for stable RESC protein-protein interactions, as well as RESC-RECC associations. Together, our data support a model in which RESC14 is necessary for assembly of editing competent RESC through recruitment of an RNP containing RESC8, GRBC and gRNA to REMC and mRNA.

In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Moreover, AGO fPAR-CLIP in Lamin A KO SHSY5Y cells revealed significantly reduced RNAi activity. Further exploration of the nuclear AGO interactome by mass spectrometry identified FAM120A, an RNA-binding protein and known interactor of AGO2. Subsequent FAM120A fPAR-CLIP, revealed that FAM120A co-binds AGO targets and that this competition reduces the RNAi activity. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, FAM120A mediated RNAi impairment, and upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.

Genome search and/or classification typically involves finding the best-match database (reference) genomes and has become increasingly challenging due to the growing number of available database genomes and the fact that traditional methods do not scale well with large databases. By combining k-mer hashing-based probabilistic data structures (i.e. ProbMinHash, SuperMinHash, Densified MinHash and SetSketch) to estimate genomic distance, with a graph based nearest neighbor search algorithm (Hierarchical Navigable Small World Graphs, or HNSW), we created a new data structure and developed an associated computer program, GSearch, that is orders of magnitude faster than alternative tools while maintaining high accuracy and low memory usage. For example, GSearch can search 8000 query genomes against all available microbial or viral genomes for their best matches (n = ∼318 000 or ∼3 000 000, respectively) within a few minutes on a personal laptop, using ∼6 GB of memory (2.5 GB via SetSketch). Notably, GSearch has an O(log(N)) time complexity and will scale well with billions of genomes based on a database splitting strategy. Further, GSearch implements a three-step search strategy depending on the degree of novelty of the query genomes to maximize specificity and sensitivity. Therefore, GSearch solves a major bottleneck of microbiome studies that require genome search and/or classification.

G-quadruplex (G4) structures play integral roles in modulating biological functions and can be regulated by small molecules. The MYC gene is critical during tumor initiation and malignant progression, in which G4 acts as an important modulation motif. Herein, we reported the MYC promoter G4 recognized by a platinum(II) compound Pt-phen. Two Pt-phen-MYC G4 complex structures in 5 mM K+ were determined by NMR. The Pt-phen first strongly binds the 3'-end of MYC G4 to form a 1:1 3'-end binding complex and then binds 5'-end to form a 2:1 complex with more Pt-phen. In the complexes, the Pt-phen molecules are well-defined and stack over four bases at the G-tetrad for a highly extensive π-π interaction, with the Pt atom aligning with the center of the G-tetrad. The flanking residues were observed to rearrange and cover on top of Pt-phen to stabilize the whole complex. We further demonstrated that Pt-phen targets G4 DNA in living cells and represses MYC gene expression in cancer cells. Our work elucidated the structural basis of ligand binding to MYC promoter G4. The platinum compound bound G4 includes multiple complexes formation, providing insights into the design of metal ligands targeting oncogene G4 DNA.

Genome segregation is a fundamental process that preserves the genetic integrity of all organisms, but the mechanisms driving genome segregation in archaea remain enigmatic. This study delved into the unknown function of SegC (SSO0033), a novel protein thought to be involved in chromosome segregation in archaea. Using fluorescence polarization DNA binding assays, we discovered the ability of SegC to bind DNA without any sequence preference. Furthermore, we determined the crystal structure of SegC at 2.8 Å resolution, revealing the multimeric configuration and forming a large positively charged surface that can bind DNA. SegC has a tertiary structure folding similar to those of the ThDP-binding fold superfamily, but SegC shares only 5-15% sequence identity with those proteins. Unexpectedly, we found that SegC has nucleotide triphosphatase (NTPase) activity. We also determined the SegC-ADP complex structure, identifying the NTP binding pocket and relative SegC residues involved in the interaction. Interestingly, images from negative-stain electron microscopy revealed that SegC forms filamentous structures in the presence of DNA and NTPs. Further, more uniform and larger SegC-filaments are observed, when SegA-ATP was added. Notably, the introduction of SegB disrupts these oligomers, with ATP being essential for regulating filament formation. These findings provide insights into the functional and structural role of SegC in archaeal chromosome segregation.

The androgen receptor (AR) is pivotal in prostate cancer (PCa) progression and represents a critical therapeutic target. AR-mediated gene regulation involves intricate interactions with nuclear proteins, with many mediating and undergoing post-translational modifications that present alternative therapeutic avenues. Through chromatin proteomics in PCa cells, we identified SUMO ligases together with nuclear receptor coregulators and pioneer transcription factors within the AR's protein network. Intriguingly, this network displayed a significant association with SUMO2/3. To elucidate the influence of SUMOylation on AR chromatin interactions and subsequent gene regulation, we inhibited SUMOylation using ML-792 (SUMOi). While androgens generally facilitated the co-occupancy of SUMO2/3 and AR on chromatin, SUMOi induced divergent effects dependent on the type of AR-binding site (ARB). SUMOi augmented AR's pioneer-like binding on inaccessible chromatin regions abundant in androgen response elements (AREs) and diminished its interaction with accessible chromatin regions sparse in AREs yet rich in pioneer transcription factor motifs. The SUMOi-impacted ARBs divergently influenced AR-regulated genes; those associated with AR-mediated activation played roles in negative regulation of cell proliferation, while those with AR-mediated repression were involved in pattern formation. In conclusion, our findings underscore the pervasive influence of SUMOylation in shaping AR's role in PCa cells, potentially unveiling new therapeutic strategies.