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Identification of PARP1-RNA binding in-vivo using PAR-CLiP Sequencing

ABSTRACT: Recent studies have implicated PARP1 in alternative splicing regulation. In addition, PARP1 has been shown to bind RNA in vivo suggesting that it is an RNA-binding protein (RBP). However, a detailed knowledge of the RNA-targets as well as the RNA binding region of PARP1 is unknown. We used Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in HeLa cells and identified a largely overlapping set ∼22142 of PARP1-RNA binding peaks mapping to mRNAs, of which ∼20484 sites are located in the intronic regions of genes. PARP1 preferentially bound RNA containing a Guanine-rich sequences. Using a bayesian model, we determined positional effects of PARP1 on exon skipping: PARP1 binding upstream and downstream to the skipped exons generally promoted exon inclusion, whereas binding on exons and closer to the skipped exon, promoted exon skipping. Finally, using truncation mutants, we narrowed the PARP1-RNA binding domain to the amino terminus region of the protein. Such studies on transcriptome-wide mapping of RNA-targets by PARP1, present the first step into understanding the role of PARP1 and RNA biogenesis. Our understanding of the role of PARPs and PAR in the transcriptional and post-transcriptional regulation of gene expression through modulation of RNA is still in the early stages. Continued identification and characterization of the functional interplay between PARPs and RNA may provide important insights into the role of PARPs in RNA regulation.

ORGANISM(S): Homo sapiens

PROVIDER: GSE95360 | GEO | 2018/02/14

REPOSITORIES: GEO

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Compound-induced NR5A1 circular RNA and aberrant multiple exon-skipping attenuate aberrant steroidogenesis

Project description:Despite the intensive search for an effective drug to ameliorate excess steroid production, there are few pharmacological options, especially for diseases as steroid-producing adrenocortical cancers. While splice-modifying-compounds have pleiotropic effects including anticancer properties, none have been tested on abnormal steroidogenesis. Using H295R adrenocortical carcinoma cells, CX-4945 induced multiple exon skipping of the NR5A1 gene, the master regulator of steroidogenesis. The resulting exon-skipped NR5A1 proteins were non-functional when added-back to NR5A1 knocked down H295R cells. This eventually suppressed steroidogenesis and induced dysfunctional autophagy with progression to ER-stress-related apoptosis. Intriguingly, a circular RNA of NR5A1 exons (circNR5A1 ex2-4 RNA) not originating from the skipped exons, was induced. Transient expression of this circNR5A1 ex2-4 RNA induced the same multiple exon-skipped isoforms of the NR5A1 gene. This potential pharmacological control of NR5A1 aberrant multiple exon-skipping and interplay with its circNR5A1 RNA gives us a novel target for treating abnormal steroidogenesis in adrenocortical carcinomas.

2022-06-24 | GSE133197 | GEO

CRISPR induced exon skipping of beta-catenin reveals unexpected tumorigenic mutants driving distinct subtypes of liver cancer

Project description:we induce exon skipping and generate a gain-of-function of an oncogene, β-catenin, using CRISPR/Cas9 in mouse liver cells. Specifically, a single guide RNA (sgRNA) targeting exon 3 of β-catenin induces exon skipping and gain-of-function of β-catenin in mouse hepatocytes. In synergy with YAPS127A, exon skipped hepatocytes gain tumorigenic ability and are thus enriched via tumor formation. Surprisingly, characterization of the exon-skipped tumors reveals two distinct subtypes with different histological features. Remarkably, ectopic expression of two representative exon-skipped β-catenin transcripts together with YAPS127A phenocopies the two histologically distinct subtypes of liver cancer. Finally, the transcriptome sequencing analysis reveal two subtypes of liver cancer and most importantly, one subtype of the exon-skipped tumor shows features of hepatoblastoma, while the other does not. This exon skipping model reveals CRISPR/Cas9 can lead to exon-skipped transcripts with in frame coding and gain-of-functions.

2021-01-01 | GSE164109 | GEO

Cotranscriptional exon skipping in the genotoxic stress response

Project description:Pre-mRNA splicing is functionally coupled to transcription, and genotoxic stresses can enhance alternative exon inclusion by affecting elongating RNA polymerase II. We report here that various genotoxic stress inducers, including camptothecin, inhibit the interaction between EWS, an RNA polymerase II-associated factor, and YB-1, a spliceosome-associated factor. This results in the cotranscriptional skipping of several exons of the MDM2 gene encoding the main p53 ubiquitin-ligase. This reversible exon skipping participates in the timely regulation of MDM2 expression, and may contribute to the accumulation of p53 during stress exposure and its rapid shut off when stress is removed. Finally, a splicing-sensitive microarray identified numerous exons that are skipped in response to camptothecin and EWS/YB-1 depletion. These data demonstrate genotoxic stress-induced alteration of the communication between the transcriptional and splicing machineries, resulting in widespread exon skipping and playing a central role in the genotoxic stress response. 6 samples of MCF7 cells exposed to different treatments were analyzed: 3 x control_6 hours; 3 x camptothecin_6 hours.

2010-06-09 | E-GEOD-21840 | biostudies-arrayexpress

Cotranscriptional exon skipping in the genotoxic stress response

2010-05-20 | GSE21840 | GEO

An Alternative Splicing Event Amplifies Evolutionary Differences Between Vertebrates

2015-09-01 | GSE69656 | GEO

Genome-wide analysis of differentially expressed genes and splicing isoforms in clear cell Renal Cell Carcinoma

Project description:In this study we performed a genome wide analysis of the entire complement of mRNAs in clear cell renal cell carcinomas (ccRCC) by means of the Affymetrix Exon Array platform. The analyses were performed both at gene and exon level. Under our parameters over 2,000 genes resulted differentially expressed, and about 250 genes resulted alternative spliced showing differential inclusion of specific cassette exons comparing tumor and non tumoral tissues. 20 total samples were used for exon array analysis: 10 ccRCC tumor sample (T) and their matched non-tumor (NT) kidney tissues samples. All exon array data were analyzed using Partek Genomic Suite 6.4 software (Partek). The robust multi-array average (RMA) algorithm was used for the gene- and exon-level intensity analyses. Data were M-oM-,M-^Altered to consider only those probe sets included in the M-bM-^@M-^\Core Meta-ProbesetM-bM-^@M-^]. Lists of genes and exons with significant variation of the expression levels were generated by using a 0.01 FDR criterion as a significant cutoff. Partek list of significant exon-level probesets (FDR corrected p-value < 0.01) and exon level ANOVA test list were used to create - by an ad hoc Python script - a database of significant probesets from Exon Array experiments. To make more stringent the analysis criteria we excluded from the final exon lists all those cases in which Exon Array revealed a significant change at gene-level. This database we created has been used to explore simple exon skipping events using as reference ASPicDB single exon skipping splicing events after mapping Affymetrix probesets on ASPicDB .gtf files. A single exon from the Partek list was defined significantly skipped only if its adjacent exons were not, by using 0.01 and 0.05 p-value criterions as a significant cutoff for the M-bM-^@M-^\skippedM-bM-^@M-^] and adjacent exons, respectively.

2013-09-06 | E-GEOD-47032 | biostudies-arrayexpress

A global regulatory mechanism for activating an exon network required for neurogenesis

Project description:The vertebrate and neural-specific SR-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells, while weakening 3' splice site recognition and contributing to exon skipping in non-neural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis. RNA-Seq was used to obtain mRNA profiles of various N2A and 293T cell lines from human and mouse, respectively, to investigate the roles of nSR100, Ptbp1 and U2af65 in alternative splicing regulation. PAR-iCLIP and iCLIP experiments followed by high throughput sequencing were conducted to obtain RNA binding profiles of nSR100, PTBP1 and U2af65.

2014-09-11 | E-GEOD-57278 | biostudies-arrayexpress

An Alternative Splicing Event Amplifies Evolutionary Differences Between Vertebrates

Project description:Alternative splicing (AS) generates extensive transcriptomic and proteomic complexity. However, the functions of species- and lineage-specific splice variants are largely unknown. Here, we show that mammalian-specific skipping of exon 9 of PTBP1 alters its splicing regulatory activities and affects the inclusion levels of numerous exons. During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program. Engineered skipping of the orthologous exon in chicken cells induces a large number of mammalian-like AS changes in PTBP1 target exons. These results thus reveal that a single exon skipping event in an RNA binding regulator directs numerous AS changes between species. The results further suggest that these changes contributed to evolutionary differences in the formation of vertebrate nervous systems. This study contains two sets of samples: (Set 1) mRNA profiling of human 293 cells subjected to four different conditions in two biological replicates: non-targetting control siRNA, PTBP1 and PTBP2 siRNA, PTBP1 and PTBP2 siRNA with overexpression of full-length human PTBP1, PTBP1 and PTBP2 siRNA with overexpression of exon-excluded human PTBP1. (Set 2) mRNA profiling of chicken DT40 cells with 3 genotypes in two bioligcal replicates: wildtype cells, cells with PTBP1 exon 8 (orthologous to human PTBP1 exon 9) deleted in one allele, and cells with PTBP1 exon 8 deleted in both alleles.

2015-09-01 | E-GEOD-69656 | biostudies-arrayexpress

SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease [miSeq]

Project description:Exon skipping technologies enable exclusion of targeted exons from mature mRNA transcripts, which has broad applications in molecular and cellular biology, medicine and biotechnology. Existing exon skipping techniques include antisense oligonucleotides, targetable nucleases and base editors, which, while effective for specific applications at some target exons, remain hindered by shortcomings preventing their broader implementation including transient effects in the case of oligonucleotides or limiting PAM motifs, sequence context preferences for deaminases, and undesirable cryptic splicing in the case of gene editing tools. To overcome these limitations, we created SPLICER, a toolbox of next-generation base editors consisting of near-PAMless Cas9 nickase variants fused with different deaminases for simultaneous editing of splice acceptor (SA) and splice donor (SD) sequences. Synchronized SA and SD editing not only improves exon skipping rates but also reduces aberrant outcomes such as cryptic splicing and intron retention. SPLICER enables editing of exon splice sites with high efficiency, including many exons refractory to splicing reprogramming by the native SpCas9 BEs. To demonstrate the therapeutic potential of SPLICER, we targeted APP exon 17, which contains the amino acid residues responsible for the formation of Aβ plaques in Alzheimer’s disease. SPLICER enabled precise and highly efficient exon skipping, which reduced the formation of Aβ42 peptides in vitro while inducing DNA editing and exon skipping in vivo within a humanized mouse model of Alzheimer’s disease. Overall, SPLICER is a widely applicable and highly efficient toolbox for exon skipping with broad therapeutic applications.

2023-12-10 | GSE246586 | GEO

SPLICER: A Highly Efficient Base Editing Toolbox That Enables in vivo Exon Skipping For Targeting Alzheimer’s Disease [RNA-seq]

2023-12-10 | GSE246587 | GEO

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