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.

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.

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.

Transcripts produced by RNA polymerase II (RNAPII) are fundamental for cellular responses to environmental changes. It is therefore no surprise that there exist multiple avenues for the regulation of this process. To explore the regulation mediated by RNAPII-interacting proteins, we used a small interfering RNA (siRNA)-based screen to systematically evaluate their influence on RNA synthesis. We identified several proteins that strongly affected RNAPII activity. We evaluated one of the top hits, SCAF1 (SR-related C-terminal domain-associated factor 1), using an auxin-inducible degradation system and sequencing approaches. In agreement with our screen results, acute depletion of SCAF1 decreased RNA synthesis, and showed an increase of Serine-2 phosphorylated-RNAPII (pS2-RNAPII). We found that the accumulation of pS2-RNAPII within the gene body occurred at GC-rich regions and was indicative of stalled RNAPII complexes. The accumulation of stalled RNAPII complexes was accompanied by reduced recruitment of initiating RNAPII, explaining the observed global decrease in transcriptional output. Furthermore, upon SCAF1 depletion, RNAPII complexes showed increased association with components of the proteasomal-degradation machinery. We concluded that in cells lacking SCAF1, RNAPII undergoes a rather interrupted passage, resulting in intervention by the proteasomal-degradation machinery to clear stalled RNAPII. While cells survive the compromised transcription caused by absence of SCAF1, further inhibition of proteasomal-degradation machinery is synthetically lethal.

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.