Saturation mutagenesis reveals manifold determinants of exon definition
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA415408 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR6202950.fastq.gz | Fastqsanger.gz | |||
| SRR6202960.fastq.gz | Fastqsanger.gz | |||
| SRR6202970.fastq.gz | Fastqsanger.gz | |||
| SRR6202951.fastq.gz | Fastqsanger.gz | |||
| SRR6202961.fastq.gz | Fastqsanger.gz |
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Project description:To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts we conducted saturation mutagenesis of a 51 nt internal exon in a 3-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high throughput genetics, 5560 minigene molecules were assayed for splicing in HEK293 cells. Over 70% of mutations produced substantial (>2X) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements only a single 15 nt stem-loop, showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1Aa, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51 nt test exon simultaneously. Surprisingly, such correlations extended to weak relative protein-sequence affinities. These myriad protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.
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Project description:To illuminate the extent and roles of exonic sequences in the splicing of human RNA transcripts, we conducted saturation mutagenesis of a 51-nt internal exon in a three-exon minigene. All possible single and tandem dinucleotide substitutions were surveyed. Using high-throughput genetics, 5560 minigene molecules were assayed for splicing in human HEK293 cells. Up to 70% of mutations produced substantial (greater than twofold) phenotypes of either increased or decreased splicing. Of all predicted secondary structural elements, only a single 15-nt stem-loop showed a strong correlation with splicing, acting negatively. The in vitro formation of exon-protein complexes between the mutant molecules and proteins associated with spliceosome formation (U2AF35, U2AF65, U1A, and U1-70K) correlated with splicing efficiencies, suggesting exon definition as the step affected by most mutations. The measured relative binding affinities of dozens of human RNA binding protein domains as reported in the CISBP-RNA database were found to correlate either positively or negatively with splicing efficiency, more than could fit on the 51-nt test exon simultaneously. The large number of these functional protein binding correlations point to a dynamic and heterogeneous population of pre-mRNA molecules, each responding to a particular collection of binding proteins.
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Project description:Saturation mutagenesis of disease-associated regulatory elements
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Project description:The Ewing’s sarcoma protein EWS belongs to the TET family (FUS/TLS, EWS, TAF15) of RNA and DNA binding proteins, implicated in DNA transcription, pre-mRNA splicing and maintenance of genomic integrity. Translocations of these genes are characteristic of particular neoplasias, including Ewing’s sarcoma. To identify physiological RNA targets of EWS, we performed in vivo cross-linking and immunoprecipitation followed by high-throughput RNA sequencing (HITS-CLIP/CLIP-Seq) in HeLa cells. Sequencing identified EWS binding sites characterized by guanosine-rich motifs in nearly 9000 genes, with particular enrichment in exonic regions near 5’ splice sites. Exon 6 of the Fas/CD95 receptor, which is alternatively spliced to generate isoforms with opposing activities in programmed cell death, was found as a prominent EWS CLIP target, as well as by chromatin-immunoprecipitation (ChIP) and functional analysis. Manipulation of EWS levels and mutation of EWS binding sites led to changes in alternative splicing consistent with EWS promoting exon 6 inclusion and leading to the synthesis of the pro-apoptotic Fas/CD95 isoform. Biochemical characterization of factors associated with FAS exon 6 are consistent with the notion that EWS binds to exonic sequences near the 5’ splice site and promotes the recruitment of U1snRNP, favoring also recognition of the upstream 3' splice site by U2AF and thus exon definition. Consistent with a role for EWS in the regulation of programmed cell death, cells depleted of EWS show decreased sensitivity to Fas-induced apoptosis. We discuss the potential implications of this novel function of EWS in Ewing’s sarcoma.
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