Hotspot exons are common targets of splicing perturbations.
Ontology highlight
ABSTRACT:
SUBMITTER: Glidden DT
PROVIDER: S-EPMC8115636 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:RNA-seq of amiloride-treated cell culture was conducted in order to understand the characteristics that make certain exons susceptible to splicing perturbations. Amiloride is a diuretic that acts by inhibiting epithelial sodium channels in the kidney. It also behaves as a non-specific kinase inhibitor, and has been shown to affect splicing in a handful of genes, presumably by altering phosphorylation of splicing factors. Two samples were sequenced (one untreated, one treated). Differential splicing was done using rMATS version 3.2.5.
2019-11-22 | GSE140786 | GEO
Project description:Hotspot exons are common targets of splicing perturbations
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Project description:Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2? (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2? is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2? binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2? regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2? binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2? protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2?. Versions of Tra2? lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2? protein.
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Project description:The spliceosomal factor TRAP150 is essential for pre-mRNA splicing in vivo and, when overexpressed, it enhances splicing efficiency. In this study, we found that TRAP150 interacted with the cleavage and polyadenylation specificity factor (CPSF) and co-fractionated with CPSF and RNA polymerase II. Moreover, TRAP150 preferentially associated with the U1 small ribonucleoprotein (snRNP). However, our data do not support a role for TRAP150 in alternative 5' splice site or exon selection or in alternative polyadenylation. Because U1 snRNP participates in premature cleavage and polyadenylation (PCPA), we tested whether TRAP150 is a cofactor in the control of PCPA. Although TRAP150 depletion had no significant effect on PCPA, overexpression of TRAP150 forced activation of a cryptic 3' splice site, yielding spliced PCPA transcripts. Mechanistic studies showed that TRAP150-activated splicing occurred in composite but not authentic terminal exons, and such an activity was enhanced by debilitation of U1 snRNP or interference with transcription elongation or termination. Together, these results indicate that TRAP150 provides an additional layer of PCPA regulation, through which it may increase the diversity of abortive RNA transcripts under conditions of compromised gene expression.
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Project description:Imperfect conservation of human pre-mRNA splice sites is necessary to produce alternative isoforms. This flexibility is combined with the precision of the message reading frame. Apart from intron-termini GU_AG and the branchpoint A, the most conserved are the exon-end guanine and +5G of the intron start. Association between these guanines cannot be explained solely by base-pairing with U1 snRNA in the early spliceosome complex. U6 succeeds U1 and pairs +5G in the pre-catalytic spliceosome, while U5 binds the exon end. Current U5 snRNA reconstructions by CryoEM cannot explain the conservation of the exon-end G. Conversely, human mutation analyses show that guanines of both exon termini can suppress splicing mutations. Our U5 hypothesis explains the mechanism of splicing precision and the role of these conserved guanines in the pre-catalytic spliceosome. We propose: (1) optimal binding register for human exons and U5-the exon junction positioned at U5Loop1 C39|C38; (2) common mechanism for base-pairing of human U5 snRNA with diverse exons and bacterial Ll.LtrB intron with new loci in retrotransposition-guided by base pair geometry; and (3) U5 plays a significant role in specific exon recognition in the pre-catalytic spliceosome. Statistical analyses showed increased U5 Watson-Crick pairs with the 5'exon in the absence of +5G at the intron start. In 5'exon positions -3 and -5, this effect is specific to U5 snRNA rather than U1 snRNA of the early spliceosome. Increased U5 Watson-Crick pairs with 3'exon position +1 coincide with substitutions of the conserved -3C at the intron 3'end. Based on mutation and X-ray evidence, we propose that -3C pairs with U2 G31 juxtaposing the branchpoint and the 3'intron end. The intron-termini pair, formed in the pre-catalytic spliceosome to be ready for transition after branching, and the early involvement of the 3'intron end ensure that the 3'exon contacts U5 in the pre-catalytic complex. We suggest that splicing precision is safeguarded cooperatively by U5, U6, and U2 snRNAs that stabilize the pre-catalytic complex by Watson-Crick base pairing. In addition, our new U5 model explains the splicing effect of exon-start +1G mutations: U5 Watson-Crick pairs with exon +2C/+3G strongly promote exon inclusion. We discuss potential applications for snRNA therapeutics and gene repair by reverse splicing.
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Project description:Many long Drosophila introns are processed by an unusual recursive strategy. The presence of ~200 adjacent splice acceptor and splice donor sites, termed ratchet points (RPs), were inferred to reflect 'zero-nucleotide exons', whose sequential processing subdivides removal of long host introns. We used CRISPR-Cas9 to disrupt several intronic RPs in Drosophila melanogaster, some of which recapitulated characteristic loss-of-function phenotypes. Unexpectedly, selective disruption of RP splice donors revealed constitutive retention of unannotated short exons. Assays using functional minigenes confirm that unannotated cryptic splice donor sites are critical for recognition of intronic RPs, demonstrating that recursive splicing involves the recognition of cryptic RP exons. This appears to be a general mechanism, because canonical, conserved splice donors are specifically enriched in a 40-80-nt window downstream of known and newly annotated intronic RPs and exhibit similar properties to a broadly expanded class of expressed RP exons. Overall, these studies unify the mechanism of Drosophila recursive splicing with that in mammals.
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Project description:Alternative splicing (AS) contributes to proteome diversity. As splicing occurs cotranscriptionally, epigenetic determinants such as DNA methylation likely play a part in regulation of AS. Previously, we have shown that DNA methylation marks exons and that a loss of DNA methylation alters splicing patterns in a genome-wide manner. To investigate the influence of DNA methylation on splicing of individual genes, we developed a method to manipulate DNA methylation in vivo in a site-specific manner using the deactivated endonuclease Cas9 fused to enzymes that methylate or demethylate DNA. We used this system to directly change the DNA methylation pattern of selected exons and introns. We demonstrated that changes in the methylation pattern of alternatively spliced exons, but not constitutively spliced exons or introns, altered inclusion levels. This is the first direct demonstration that DNA methylation of exon-encoding regions is directly involved in regulation of AS.
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Project description:Exonization of Alu elements creates primate-specific genomic diversity. Here we combine bioinformatic and experimental methodologies to reconstruct the molecular changes leading to exon selection. Our analyses revealed an intricate network involved in Alu exonization. A typical Alu element contains multiple sites with the potential to serve as 5' splice sites (5'ss). First, we demonstrated the role of 5'ss strength in controlling exonization events. Second, we found that a cryptic 5'ss enhances the selection of a more upstream site and demonstrate that this is mediated by binding of U1 snRNA to the cryptic splice site, challenging the traditional role attributed to U1 snRNA of binding the 5'ss only. Third, we used a simple algorithm to identify specific sequences that determine splice site selection within specific Alu exons. Finally, by inserting identical exons within different sequences, we demonstrated the importance of flanking genomic sequences in determining whether an Alu exon will undergo exonization. Overall, our results demonstrate the complex interplay between at least four interacting layers that affect Alu exonization. These results shed light on the mechanism through which Alu elements enrich the primate transcriptome and allow a better understanding of the exonization process in general.
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Project description:Alternative processing of pre-mRNA plays an important role in protein diversity and biological function. Previous studies on alternative splicing (AS) often focused on the spatial patterns of protein isoforms across different tissues. Here we studied dynamic usage of AS across time, during murine retina development. Over 7000 exons showed dynamical changes in splicing, with differential splicing events occurring more frequently in early development. The overall splicing patterns for exclusive and inclusive exons show symmetric trends and genes with symmetric splicing patterns that tend to have similar biological functions. Furthermore, we observed that within the retina, retina-enriched genes that are preferentially expressed at the adult stage tend to have more dynamically spliced exons compared to other genes, suggesting that genes maintaining retina homeostasis also play an important role in development via a series of AS events. Interestingly, the transcriptomes of retina-enriched genes largely reflect the retinal developmental process. Finally, we identified a number of candidate cis-regulatory elements for retinal AS by analyzing the relative occurrence of sequence motifs in exons or flanking introns. The occurrence of predicted regulatory elements showed strong correlation with the expression level of known RNA binding proteins, suggesting the high quality of the identified cis-regulatory elements.
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