Nucleic Acids Research最新文献

Ewing sarcomas (ESs) are biologically aggressive tumours of bone and soft tissues caused by chromosomal translocations yielding in-frame fusion proteins driving the neoplastic transformation. The DNA/RNA helicase DHX9 is an important regulator of cellular processes often deregulated in cancer. Using transcriptome profiling, our study reveals cancer-relevant genes whose splicing is modulated by DHX9. Immunodepletion experiments demonstrate that DHX9 impacts on the recruitment of U2 small nuclear RNP (snRNP) onto the pre-mRNA. Analysis of structure and sequence features of DHX9 target exons reveal that DHX9-sensitive exons display shorter flanking introns and contain HNRNPC and TIA1 consensus motifs. A prominent target of DHX9 is exon 11 in the Cortactin (CTTN) gene, which is alternatively spliced to generate isoforms with different activities in cell migration and tumour invasion. Alternative inclusion of the exon 11 in CTTN gene is one of the most recurrent isoform switches in multiple cancer types, thus highlighting the pivotal role of DHX9 in defining the tumour phenotype. Biochemical analyses reveal that DHX9 binding promotes the recruitment of U2snRNP, SF3B1, and SF3A2 to the splice sites flanking exon 11. These findings uncover a new role of DHX9 in the control of co-transcriptional splicing in ES, which may represent a new druggable target to counteract ES malignancy.

Rates of transcription elongation vary within and across eukaryotic gene bodies. Here, we introduce new methods for predicting elongation rates from nascent RNA sequencing data. First, we devise a probabilistic model that predicts nucleotide-specific elongation rates as a generalized linear function of nearby genomic and epigenomic features. We validate this model with simulations and apply it to public PRO-seq (Precision Run-On Sequencing) and epigenomic data for four cell types, finding that reductions in local elongation rate are associated with cytosine nucleotides, DNA methylation, splice sites, RNA stem-loops, CTCF (CCCTC-binding factor) binding sites, and several histone marks, including H3K36me3 and H4K20me1. By contrast, increases in local elongation rate are associated with thymines, A+T-rich and low-complexity sequences, and H3K79me2 marks. We then introduce a convolutional neural network that improves our local rate predictions. Our analysis is the first to permit genome-wide predictions of relative nucleotide-specific elongation rates.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by loss-of-function mutations in the MECP2 gene, resulting in diverse cellular dysfunctions. Here, we investigated the role of the long noncoding RNA (lncRNA) NEAT1 in the context of MeCP2 deficiency using human neural cells and RTT patient samples. Through single-cell RNA sequencing and molecular analyses, we found that NEAT1 is markedly downregulated in MECP2 knockout (KO) cells at various stages of neural differentiation. NEAT1 downregulation correlated with aberrant activation of the mTOR pathway, abnormal protein metabolism, and dysregulated autophagy, contributing to the accumulation of protein aggregates and impaired mitochondrial function. Reactivation of NEAT1 in MECP2-KO cells rescued these phenotypes, indicating its critical role downstream of MECP2. Furthermore, direct RNA-RNA interaction was revealed as the key process for NEAT1 influence on autophagy genes, leading to altered subcellular localization of specific autophagy-related messenger RNAs and impaired biogenesis of autophagic complexes. Importantly, NEAT1 restoration rescued the morphological defects observed in MECP2-KO neurons, highlighting its crucial role in neuronal maturation. Overall, our findings elucidate lncRNA NEAT1 as a key mediator of MeCP2 function, regulating essential pathways involved in protein metabolism, autophagy, and neuronal morphology.

During meiosis, the number and distribution of crossovers (COs) must be precisely regulated through CO assurance and interference to prevent chromosome missegregation and genomic instability in the progeny. Here we show that this regulation of COs depends on a disordered and conserved domain within the synaptonemal complex (SC). This domain is located at the C-terminus of the central element protein SYP-4 in Caenorhabditis elegans. While not necessary for synapsis, the C-terminus of SYP-4 is crucial for both CO assurance and interference. Although the SYP-4 C-terminus contains many potential phosphorylation sites, we found that phosphorylation is not the primary regulator of CO events. Instead, we discovered that nine conserved phenylalanines are required to recruit a pro-CO factor predicted to be an E3 ligase and regulate the physical properties of the SC. We propose that this conserved and disordered domain plays a crucial role in maintaining the SC in a state that allows transmitting signals to regulate CO formation. While the underlying mechanisms remain to be fully understood, our findings align with existing models suggesting that the SC plays a critical role in determining the number and distribution of COs along chromosomes, thereby safeguarding the genome for future generations.

Lung cancer sequencing efforts have uncovered mutational signatures that are attributed to exposure to the cigarette smoke carcinogen benzo[a]pyrene. Benzo[a]pyrene metabolizes in cells to benzo[a]pyrene diol epoxide (BPDE) and reacts with guanine nucleotides to form bulky BPDE adducts. These DNA adducts block transcription and replication, compromising cell function and survival, and are repaired in human cells by the nucleotide excision repair pathway. Here, we applied high-resolution genomic assays to measure BPDE-induced damage formation and mutagenesis in human cells. We integrated the new damage and mutagenesis data with previous repair, DNA methylation, RNA expression, DNA replication, and chromatin component measurements in the same cell lines, along with lung cancer mutagenesis data. BPDE damage formation is significantly enhanced by DNA methylation and in accessible chromatin regions, including transcribed and early-replicating regions. Binding of transcription factors is associated primarily with reduced, but also enhanced damage formation, depending on the factor. While DNA methylation does not appear to influence repair efficiency, this repair was significantly elevated in accessible chromatin regions, which accumulated fewer mutations. Thus, when damage and repair drive mutagenesis in opposing directions, the final mutational patterns appear to be dictated by the efficiency of repair rather than the frequency of underlying damages.

Plasmid-encoded toxin-antitoxin (TA) systems are known for their role in plasmid maintenance via post-segregational killing. Here, we identified an inactive type II TA system, MtvT/MtvA (MtvTA), encoded on the conjugative plasmid pPAD8 from the clinical Pseudomonas aeruginosa strain PAD8. Despite its annotation as a toxin, MtvT exhibited no detectable toxicity in our assays. Interestingly, the deletion of the MtvTA significantly increased the transfer efficiency of pPAD8 from PAD8 to P. aeruginosa strain PAO1. Functional assays revealed that the MtvTA complex negatively regulates plasmid transfer by binding to the promoters of dot/icm system genes. In addition, pPAD8ΔmtvTA attenuated the pathogenicity of the host strain compared to pPAD8, highlighting a regulatory role for MtvTA in virulence. Mechanistically, the MtvTA complex positively regulates the type III and type VI secretion systems and pyocyanin biosynthesis by directly binding to the promoters of exsA and rsmY/rsmZ and indirectly influencing lasI expression, respectively. These findings provide new insights into the regulatory roles of an inactive plasmid-encoded TA system, expanding our understanding of the interplay between plasmids and their bacterial hosts.

The alternative lengthening of telomeres (ALT) pathway is a telomerase-independent mechanism for immortalization in cancer cells and is commonly activated in low-grade and high-grade glioma, as well as osteosarcoma. The ALT pathway can be activated under various conditions and has often been shown to include mutational loss of ATRX. However, this is insufficient in isolation and so other cellular event must also be implicated. It has been shown that excessive accumulation of DNA:RNA hybrid structures (R-loops) and/or formation of DNA-protein crosslinks (DPCs) can be other important driving factors. The underlying cellular events leading to R-loop and DPC formation in ALT cancer cells to date remain unclear. Here, we demonstrate that excessive cellular reactive oxygen species (ROS) is an important causative factor in the evolution of ALT-telomere maintenance in ATRX-deficient glioma. We identified three sources of elevated ROS in ALT-positive gliomas: co-mutation of SETD2, downregulation of DRG2, and hypoxic tumour microenvironment. We demonstrate that elevated ROS leads to accumulation of R-loops and, crucially, resolution of R-loops by the enzyme RNase H1 prevents ALT pathway activity in cells exposed to elevated ROS. Further, we found a possible causal link between the formation of R-loops and the accumulation of DPCs, in particular, formation of TOP1 complexes covalently linked to DNA (Top1cc). We also demonstrate that elevation of ROS can trigger over-activity of the ALT pathway in osteosarcoma and glioma cell lines, resulting in excessive DNA damage and cell death. This work presents important mechanistic insights into the endogenous origin of excessive R-loops and DPCs in ALT-positive cancers, as well as highlighting potential novel therapeutic approaches in these difficult-to-treat cancer types.

Genetic screens using CRISPR (Clustered Regularly Interspaced Palindromic Repeats) provide valuable information about gene function. Nearly all pooled screening technologies rely on the cell to link genotype to phenotype, making it challenging to assay mechanistically informative, biochemically defined phenotypes. Here, we present CRISPuRe-seq (CRISPR PuRification), a novel pooled screening strategy that expands the universe of accessible phenotypes through the purification of ribonucleoprotein complexes that link genotypes to expressed RNA barcodes. While screening for regulators of the integrated stress response (ISR), we serendipitously discovered that the ISR represses transfer RNA (tRNA) production under conditions of reduced protein synthesis. This regulation is mediated through inhibition of mTORC1 and corresponding activation of the RNA polymerase III inhibitor MAF1. These data demonstrate that coherent downregulation of tRNA expression and protein synthesis is achieved through cross-talk between the ISR and mTOR, two master integrators of cell state.

Nucleic acid ADP-ribosylation and its associated enzymes involved in catalysis and hydrolysis are widespread among all kingdoms of life. Yet, its roles in mammalian and bacterial physiology including inter-/intraspecies conflicts are currently underexplored. Recently, several examples of enzymatic systems for RNA ADP-ribosylation have been identified, showing that all major types of RNA species, including messenger RNA, ribosomal RNA, and transfer RNA, can be targeted by ADP-ribosyltransferases (ARTs) which attach ADP-ribose modifications either to nucleobases, the backbone ribose, or phosphate ends. Yet little is known about the reversibility of RNA ADP-ribosylation by ADP-ribosylhydrolases belonging to the macrodomain, ARH, or NADAR superfamilies. Here, we characterize the hydrolytic activity of ADP-ribosylhydrolases on RNA species ADP-ribosylated by mammalian and bacterial ARTs. We demonstrate that NADAR ADP-ribosylhydrolases are the only hydrolase family able to reverse guanosine RNA base ADP-ribosylation while they are inactive on phosphate-end RNA ADP-ribosylation. Furthermore, we reveal that macrodomain-containing PARG enzymes are the only hydrolase type with the ability for specific and efficient reversal of 2'-hydroxyl group RNA ADP-ribosylation catalysed by Pseudomonas aeruginosa effector toxin RhsP2. Moreover, using the RhsP2/bacterial PARG system as an example, we demonstrate that PARG enzymes can act as protective immunity enzymes against antibacterial RNA-targeting ART toxins.

Proliferating cell nuclear antigen (PCNA) is essential for the faithful duplication of eukaryotic genomes. PCNA also orchestrates events necessary to address threats to genomic integrity, such as the DNA damage tolerance (DDT) response, a mechanism by which eukaryotic cells bypass replication-blocking lesions to maintain replisome stability. DDT is regulated by the ubiquitylation of PCNA and the consequent recruitment of specialized polymerases that ensure replication continuity. We have recently described that the deubiquitylases Ubp10 and Ubp12 modulate DDT events by reverting the ubiquitylation of PCNA in Saccharomyces cerevisiae. This study identifies Ubp1 as a novel PCNA deubiquitylase that cooperates with Ubp10 and Ubp12 in the regulation of DDT during DNA replication. Ubp1, previously known as a cytoplasmic protein, also localizes to the nucleus, where it associates with DNA replication forks. Additionally, Ubp1 interacts with and deubiquitylates PCNA. Here, we provide evidence that Ubp1 collaborates with Ubp10 and Ubp12 to facilitate DNA replication by efficiently reverting PCNAK164 ubiquitylation at replication forks under conditions free from exogenous perturbations. Consequently, the deletion of UBP1, UBP10, and UBP12 leads to persistent ubiquitylation of PCNAK164 and a marked delay in S phase progression.