Conserved intronic secondary structures with concealed branch sites regulate alternative splicing of poison exons
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA925017 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:Alternative splicing (AS) generates large amounts of RNA isoforms and dramatically increases the complexities of transcriptomes and proteomes. However, it remains unclear how RNA structures contribute to AS regulation. Here, we systematically search transcriptomes for secondary structures with concealed branch-sites (BSs) in the alternatively spliced introns and predict thousands from six organisms, of which many are evolutionarily conserved. Intriguingly, a highly conserved stem-loop structure with concealed BSs is found in animal SF3B3 genes and colocalizes with a downstream poison exon (PE). Destabilization of this structure allows increased usage of the branch sites and results in enhanced PE inclusion in both human and Drosophila cells, leading to decreased expression of SF3B3. Through an RNAi screen of 28 RNA-binding proteins, we find that this stem-loop structure is sensitive to U2 and U2-associated proteins. Furthermore, we find that SF3B3 also facilitates DNA repair and protects genome stability by enhancing interaction between ERCC6/CSB and arrested RNA polymerase II. Importantly, both Drosophila and human cells with the secondary structure mutated by genome editing exhibit altered DNA repair in vivo. This study provides a novel and common mechanism for AS regulation of PEs and reveals a physiological function of SF3B3 in DNA repair.
2024-06-12 | GSE223117 | GEO
Project description:Both canonical and alternative splicing of RNAs is governed by intronic sequence elements and produces transient lariat structures fastened by branch-points within introns. To map precisely the location of branch-points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes, and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon skipping branch-points. LaSSO thus provides an effective, accurate and unbiased approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated to RNA-seq pipelines. Interrogation of the S. pombe transcriptome using rRNA depleted strand specific RNA sequencing (Illumina HiSeq 2000) in wild type and dbr1.M-NM-^T cultures. A total of 4 samples were analyzed: two biological repeates of wild-type strain and two biological repeats of dbr1.M-NM-^T
2014-03-18 | E-GEOD-50246 | biostudies-arrayexpress
Project description:Both canonical and alternative splicing of RNAs is governed by intronic sequence elements and produces transient lariat structures fastened by branch-points within introns. To map precisely the location of branch-points on a genomic scale, we developed LaSSO (Lariat Sequence Site Origin), a data-driven algorithm which utilizes RNA-seq data. Using fission yeast cells lacking the debranching enzyme Dbr1, LaSSO not only accurately identified canonical splicing events, but also pinpointed novel, but rare, exon-skipping events, which may reflect aberrantly spliced transcripts. Compromised intron turnover perturbed gene regulation at multiple levels, including splicing and protein translation. Notably, Dbr1 function was also critical for the expression of mitochondrial genes, and for the processing of self-spliced mitochondrial introns. LaSSO showed better sensitivity and accuracy than algorithms used for computational branch-point prediction or for empirical branch-point determination. Even when applied to a human data set acquired in the presence of debranching activity, LaSSO identified both canonical and exon skipping branch-points. LaSSO thus provides an effective, accurate and unbiased approach for defining high-resolution maps of branch-site sequences and intronic elements on a genomic scale. LaSSO should be useful to validate introns and uncover branch-point sequences in any eukaryote, and it could be integrated to RNA-seq pipelines.
2014-03-18 | GSE50246 | GEO
Project description:RNA isoform screens reveal the essentiality and tumor suppressor activity of ultraconserved poison exons
| PRJNA494172 | ENA
Project description:Systematic mutagenesis has revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, suggesting that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between sequence variation and exon inclusion across the genome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across the genome but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.
2020-06-29 | GSE151942 | GEO
Project description:Somatic SF3b1 mutations occur in many cancers, and the highly conserved H662 residue is one of the most frequently mutated sites. To address their mechanism on splicing alteration and effects on development, we constructed mutant strains at the corresponding residue H698 in Drosophila using CRISPR-Cas9 system. Two mutations, H698D and H698R, were selected due to their presence in patients and the significantly opposite charges. Both the sf3b1-H698D and –H698R mutant flies exhibit developmental defects including less egg-laying, decreased hatching rates, delayed morphogenesis and shorter lifespan. Interestingly, the H698D mutant has decreased resistance to fungi infection, while the H698R mutant has impaired climbing ability. Further RNA-seq data finds altered expression of immunity response genes and changed alternative splicing of muscle and neural-related genes in the two mutants respectively. Lariat sequencing reveal that the sf3b1-H662 mutations cause aberrant selection of multiple intronic branch sites and the H698R mutant prefers to use upstream branch sites in the alternative splicing changed events. This study provides in vivo evidence from Drosophila to elucidate how the highly conserved SF3b1 cancer mutations alter splicing and their consequences in the development and immune systems.
2021-10-08 | GSE171002 | GEO
Project description:Paired gRNA/Cas9 delivery facilitates the removal of alternative exons and reveals splicing dependent phenotypes.
2019-11-19 | GSE120703 | GEO
Project description:Regulation and functionality of species-specific alternative splicing has remained enigmatic for many years. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific Camk2β alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strength during evolution globally renders constitutive exons alternative, thus providing a paradigm for cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9 we introduced the weaker human branch point into the mouse genome, resulting in human-like CAMK2B splicing in the brain of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled, species-specific alternative splicing with a fundamental function in learning and memory.
2022-12-19 | PXD035346 | Pride
Project description:Poison frogs sequester chemical defenses from their diet of leaf litter arthropods for defense against predation. Little is known about the physiological adaptations that confer this unusual bioaccumulation ability. We conducted an alkaloid-feeding experiment with the Diablito poison frog (Oophaga sylvatica) to determine how quickly alkaloids are accumulated and how toxins modify frog physiology using quantitative proteomics. Diablito frogs rapidly accumulated the alkaloid decahydroquinoline within four days, and dietary alkaloid exposure modified protein abundance in the intestines, liver, and skin. Many proteins that increased in abundance with toxin accumulation are plasma glycoproteins, including the complement system and the toxin-binding protein saxiphilin. Other protein classes that change in abundance with toxin accumulation are membrane proteins involved in small molecule transport and metabolism. Overall, this work shows poison frogs can rapidly accumulate alkaloids, which alter carrier protein abundance, initiate an immune response, and alter small molecule transport and metabolism dynamics across tissues
2021-01-25 | PXD021216 | Pride
Project description:Regulation and functionality of species-specific alternative splicing has remained enigmatic for many years. Calcium/calmodulin-dependent protein kinase IIβ (CaMKIIβ) is expressed in several splice variants and plays a key role in learning and memory. Here, we identify and characterize several primate-specific CAMK2B splice isoforms, which show altered kinetic properties and changes in substrate specificity. Furthermore, we demonstrate that primate-specific Camk2β alternative splicing is achieved through branch point weakening during evolution. We show that reducing branch point and splice site strength during evolution globally renders constitutive exons alternative, thus providing a paradigm for cis-directed species-specific alternative splicing regulation. Using CRISPR/Cas9 we introduced the weaker human branch point into the mouse genome, resulting in human-like CAMK2B splicing in the brain of mutant mice. We observe a strong impairment of long-term potentiation in CA3-CA1 synapses of mutant mice, thus connecting branch point-controlled, species-specific alternative splicing with a fundamental function in learning and memory.
2022-12-02 | GSE208181 | GEO