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P53 exon 11 mutants generated by CRISPR/Cas9 result in aberrant alternative splicing.

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE131416 | GEO | 2020/05/01

REPOSITORIES: GEO

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Project description:Alternative pre-messenger RNA splicing impacts development, physiology, and disease, but its regulation in humans is not well understood, partially due to the limited scale to which the expression of specific splicing events has been measured. We generated the first genome-scale expression compendium of human alternative splicing events using custom whole-transcript microarrays monitoring expression of 24,426 mutually exclusive alternative splicing event pairs in 48 diverse human samples. Over 11,700 genes and 9,500 splicing events were differentially expressed, providing a rich resource for studying splicing regulation. An unbiased, systematic screen of 21,760 4-mer to 7-mer words for cis-regulatory motifs identified 143 RNA 'words' enriched near regulated cassette exons, including six clusters of motifs represented by UCUCU, UGCAUG, UGCU, UGUGU, UUUU, and AGGG, which map to trans-acting regulators PTB, Fox, Muscleblind, CELF/CUG-BP, TIA-1, and hnRNP F/H, respectively. Each cluster showed a distinct pattern of genomic location and tissue specificity. For example, UCUCU occurs 110 to 35 nucleotides preceding cassette exons upregulated in brain and striated muscle but depleted in other tissues. UCUCU and UGCAUG appear to have similar function but independent action, occurring 5' and 3', respectively, of 33% of the cassette exons upregulated in skeletal muscle but co-occurring for only 2%. Keywords: multiple tissue comparison PolyA+ purified RNA pooled from multiple donors of a single human tissue type (e.g. cerebellum) were amplified with random primers and hybridized on a two-color ink-jet oligonucletodie microarray (17 array set) against a common reference pool, comprising ~20 normal adult tissue pools, on custom microarray patterns containing probes to monitor every exon and exon-exon junction in transcript databases, patent databases, and predicted from mouse transcripts. Data were analyzed for gene expression (the average of multiple probes), exon and junction expression, and splice form proportionality (see paper).

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P53 exon 11 mutants generated by CRISPR/Cas9 result in aberrant alternative splicing.

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