P53 exon 11 mutants generated by CRISPR/Cas9 result in aberrant alternative splicing.
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA543503 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9082130.fastq.gz | Fastqsanger.gz | |||
| SRR9082140.fastq.gz | Fastqsanger.gz | |||
| SRR9082131.fastq.gz | Fastqsanger.gz | |||
| SRR9082141.fastq.gz | Fastqsanger.gz | |||
| SRR9082132.fastq.gz | Fastqsanger.gz |
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Project description:The Tp53 gene encodes the famous tumor suppressor protein p53. This gene is encoded in 11 exons and various alternative spliced versions of the transcripts have been detected. Here we generated targeted mutations in human colon cancer cell line HCT116 using CRISPR/Cas9 genome editing tools. The mutations were targeted to the 5' end of Tp53 gene exon 11. CRISPR/Cas9 transfected cells were allowed to repair double stranded breaks by non-homologous end joining. Mutant cell pools were single cell cloned and analyzed by Sanger DNA sequencing. Three mutant clones, mut4, mut5 and mut9 were found to contain biallelic homozygous mutations. Poly adenylated RNA was isolated from mutant cell pools was isolated and sequenced by Illumina RNASeq in triplicate. We find that exon 11 mutations result in the alternative splicing of earlier exons and cause the expression of the p53beta alternative splice product. This C-terminally deleted protein has been associated with the induction of senescence. We thus identify an exonic splicing silencer in exon 11 of the Tp53 gene which normally suppressed alternative splicing events that encode the p53beta variant. This sequence and the factors that associate with it are prime candidates for targeting, which may induce p53beta splicing and the induction of senescence in tumors.
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Project description:Compound-induced NR5A1 circular RNA and aberrant multiple exon-skipping attenuate aberrant steroidogenesis
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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.
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Project description:Splicing alterations are very common in cancer and might affect disease initiation and progression. In several cancers, mutations in splicing factor genes are responsible for aberrant splicing. We show that certain aggressive cancers, such as pediatric T-cell acute lymphoblastic leukemia (T-ALL), have an alternative mechanism for aberrant splicing that involves post-translational regulation of the splicing machinery via deubiquitination. We initially demonstrated that T-ALL presents with extensive exon skipping events affecting proteasomal transcripts, cell cycle and epigenetic regulators. Performing an unbiased genetic screen study for RNA binding proteins, we showed that the serine/arginine-rich splicing factor (SRSF) proteins, controlling exon skipping are critical for T-ALL survival. In our effort to dissect mechanisms of aberrant regulation of SRSF proteins, we focused on the pro-oncogenic deubiquitinase ubiquitin-specific peptidase 7 (USP7) to show it regulates the levels of the SRSF proteins at the post-translational level via active deubiquitination. We further demonstrated that USP7 inhibitors can change the splicing landscape and block T-ALL growth. Our drug studies show that decrease of the levels of SRSF proteins splicing inhibitors sensitize cells to the clinically used splicing inhibitor H3B-8800. Driven by our molecular findings on aberrant splicing of proteasomal transcripts, we demonstrate that H3B-8800 could act synergistically with proteasome inhibitors, paving the way for new therapeutic schemes in pediatric leukemia. Collectively this study provides the proof-of-principle for regulation of splicing factors independently of mutations and via deubiquitination and suggests new therapeutic modalities and combinations in cancer.
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Project description:Recurrent mutations in RNA splicing factors SF3B1, U2AF1, and SRSF2 have been reported in hematologic cancers including myelodysplastic syndromes (MDS) and chronic lymphocytic leukemia (CLL). However, SF3B1 is the only splicing associated gene to be found mutated in CLL and has been shown to induce aberrant splicing. To investigate if any other genomic aberration caused similar transcriptome changes, we clustered RNASeq samples based on an alternative 3’ splice site (ss) pattern previously identified in SF3B1-mutant CLL patients. Out of 215 samples, we identified 37 (17%) with alternative 3’ ss usage, the majority of which harbored known SF3B1 hotspot mutations. Interestingly, 3 patient samples carried previously unreported in-frame deletions in SF3B1 around K700, the most frequent mutation hotspot. To study the functional effects of these deletions, we used various minigenes demonstrating that recognition of canonical 3’ ss and alternative branchsite are required for aberrant splicing, as observed for SF3B1 p.K700E. The common mechanism of action of these deletions and substitutions result in similar sensitivity of primary cells towards splicing inhibitor E7107. Altogether, these data demonstrate that novel SF3B1 in-frame deletion events identified in CLL result in aberrant splicing, a common biomarker in spliceosome-mutant cancers.
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Project description:MSI (Microsatellite Instability) colorectal cancer (CRC) show improved survival, are less prone to metastasis and show poor response to chemotherapy (compared to MSS tumors). The underlying reasons for these characteristics are still not understood and no specific therapeutic approach for MSI colon tumours (15% of CRC overall) has yet been developed.
The MSI process is oncogenic when it affects DNA repeat sequences that have a functional role, e.g. Small Coding Repeats (SCR). MSI also frequently affects Long Non-Coding Repeats (LNCR) in tumour DNA. In contrast to SCR, only a few LNCR are endowed with biological activity. Consequently, this area has received very little attention. Our group recently identified HSP110 mutant chaperone protein in MSI CRC that was generated by somatic deletion of a LNCR. Of interest, HSP110 mutant (due to exon skipping) have anti-oncogenic properties and the survival of MSI CRC patients receiving chemotherapy is positively associated with HSP110 mutations in tumour DNA.
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1. To identify exon/intron sites affected by aberrant splicing events due to MSI in CRC . All RNASeq data will be exploited to identify recurrent splicing aberrations (mostly exon skipping) that occur specifically in MSI colon tumours;
2. To investigate for possible functional links between MSI and any detected aberrant splicing events . All specific aberrant splicing events detected by RNAseq in MSI CRC samples will be first confirmed (quantitative RT-PCR) in order to eliminate false positive cases. For validated exon candidates, the allelic profiles of adjacent intronic LNCR will be analysed (PCR and fluorescence genotyping) in CRC cell lines and primary tumours (MSI and MSS), as well as in matching normal mucosa samples in order to assess their polymorphic status;
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2023-03-11 | PXD037448 | Pride
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