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Synthetic introns enable mutation-dependent targeting of cancer cells

ABSTRACT: Many cancers carry recurrent change-of-function mutations in RNA splicing factor genes, which induce sequence-specific changes in RNA splicing. Here, we describe a method to harness this change in RNA splicing activity to drive splicing factor mutation-dependent gene expression in cancers and selectively eliminate these tumors. We engineered synthetic introns which were efficiently spliced in leukemia and breast epithelial cells bearing the most common SF3B1 mutations, but unspliced in wild-type cells—and vice versa—to yield mutation-dependent protein production. A massively parallel screen of 8,881 distinct introns delineated ideal intronic size, mapped essential sequence elements, and revealed the basis of mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus thymidine kinase and subsequent ganciclovir-mediated elimination of leukemia and breast epithelial cells bearing SF3B1 mutations, while leaving wild-type cells unaffected. This approach significantly decreased the growth of otherwise lethal leukemia xenografts and correspondingly improved host survival. The modular, compact, and specific nature of synthetic introns thereby provide a means to exploit cancer-specific changes in RNA splicing for genotype-dependent gene expression and gene therapy.

ORGANISM(S): Homo sapiens

PROVIDER: GSE163217 | GEO | 2021/12/30

REPOSITORIES: GEO

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GPATCH8 modulates mutant SF3B1 mis-splicing and pathogenicity in hematologic malignancies

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2024-04-10 | PXD051128 | Pride

H3B-8800, a novel oral splicing modulator, induces lethality in spliceosome mutant cancers [K562]

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H3B-8800, a novel oral splicing modulator, induces lethality in spliceosome mutant cancers [Nalm-6]

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Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [TRIBE-seq]

Project description:Mutations in the RNA splicing factor SF3B1 are common across hematologic and solid cancers and result in widespread alterations in splicing but therapeutic means to correct this mis-splicing do not exist. Here we utilize synthetic introns uniquely responsive to mutant SF3B1 to identify trans factors required for aberrant mutant SF3B1 splicing activity. This revealed the G-patch domain containing protein GPATCH8 as required for mutant SF3B1-induced splicing alterations and impaired hematopoiesis. GPATCH8 is involved in quality control of branchpoint selection, interacts with the RNA helicase DHX15, and functionally opposes SUGP1, a G-patch protein recently implicated in SF3B1 mutant diseases. Silencing of GPATCH8 corrected one-third of mutant SF3B1 splicing defects and was sufficient to improve hematopoiesis in SF3B1 mutant mouse and human cells. These data identify GPATCH8 as a novel splicing factor required for mis-splicing by mutant SF3B1 and the therapeutic impact of correcting aberrant splicing in SF3B1 mutant cancers.

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Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations [RNA-Seq 2]

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Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations

Project description:Synthetic Introns Identify GPATCH8 as Required for Mis-splicing by SF3B1 Mutations

| PRJNA1011318 | ENA

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Project description:Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. Part of the explanation probably lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicated that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses revealed that SF3B1 is specifically bound to nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affected the splicing of these exons similarly to inhibition of SF3B1 expression. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure. MNase-seq on Input and SF3B1 pull-down, mRNA-seq on control and SF3B1 si-RNA treated cells as well as on TSA (Trichostatin A) treated and untreated cells.

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Transcriptome analysis of chronic lymphoid leukemia reveals isoform regulation associated with mutations in SF3B1

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