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.
2017-11-27 | GSE105785 | GEO
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 majority of common variants associated with common diseases, as well as an unknown proportion of causal mutations for rare diseases, fall in noncoding regions of the genome. Although catalogs of noncoding regulatory elements are steadily improving, we have a limited understanding of the functional effects of mutations within them. Here, we performed saturation mutagenesis in conjunction with massively parallel reporter assays on 20 disease-associated gene promoters and enhancers, generating functional measurements for over 30,000 single nucleotide substitution and deletion mutations. We find that the density of putative transcription factor binding sites varies widely between regulatory elements, as does the extent to which evolutionary conservation or various integrative scores predict functional effects. These data provide a powerful resource for interpreting the pathogenicity of clinically observed mutations in these disease-associated regulatory elements, and also comprise a gold-standard dataset for the further development of algorithms that aim to predict the regulatory effects of noncoding mutations.
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Project description:Long introns with short exons in vertebrate genes are thought to require spliceosome assembly across exons (exon definition), rather than introns, thereby requiring transcription of an exon to splice an upstream intron. Here, we developed CoLa-seq (co-transcriptional lariat sequencing) to investigate the timing and determinants of co-transcriptional splicing genome wide. Unexpectedly, 90% of all introns, including long introns, can splice before transcription of a downstream exon, indicating that exon definition is not obligatory for most human introns. Still, splicing timing varies dramatically across introns, and various genetic elements determine this variation. Strong U2AF2 binding to the polypyrimidine tract predicts early splicing, explaining exon definition-independent splicing. Together, our findings question the essentiality of exon definition and reveal features beyond intron and exon length that are determinative for splicing timing.
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Project description:We employed saturation genome editing (SGE) to assess the functional consequences of synonymous, missense, and nonsense variants across KARS1 exon 2. Deep DNA sequencing of fixed-amplicon PCR products targeting the endogenous KARS1 Exon 2 locus was used to determine variant frequencies as part of a larger study to identify a set of reproducible enrichment scores indicating effects of variants on KARS1 function.
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2016-05-01 | E-MTAB-4143 | biostudies-arrayexpress
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.
2014-05-21 | GSE48065 | GEO
Project description:Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon-exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity either from an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wildtype cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon de-silencing in EJC-depleted Drosophila ovaries. We propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. Our observations provide a powerful basis to dissect the molecular events that underlie the role of the EJC in splicing.
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