Wide spread and efficient co-transcriptional splicing facilitated by multiple exons
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA528286 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR8757100_1.fastq.gz | Fastqsanger.gz | |||
| SRR8757100_2.fastq.gz | Fastqsanger.gz | |||
| SRR8757110_1.fastq.gz | Fastqsanger.gz | |||
| SRR8757110_2.fastq.gz | Fastqsanger.gz | |||
| SRR8757101_1.fastq.gz | Fastqsanger.gz |
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Project description:Pre-mRNA splicing is important for gene expression in most eukaryotic organisms. Regulation occurs during pre-mRNA splicing greatly expanded the transcriptome complexity. Recent studies from Mammals, Drosophila and Yeast showed that the majority of introns are spliced co-transcriptionally. However, in plant the nature of co-transcriptionally splicing (CTS) and its regulation is still largely unknown. Here, through sequencing the chromatin-bound RNA (CB-RNA-seq), we studied the feature of CTS in Arabidopsis. We found CTS is widespread in Arabidopsis seedlings and a large proportion of alternative splicing events are determined co-transcriptionally. We found the CTS efficiency correlate with gene expression level, chromatin landscape and most surprisingly, the number of intron/exon of individual genes, whilst independent of gene length. In combination with iCLIP analysis, we found splicing regulator RZ-1B/1C promotes efficient CTS of thousands genes involving direct binding mainly to the exonic sequences. Interestingly, for many cases, the splicing promotion activity of RZ-1C does not associate with its binding to the regions immediately adjacent to the regulated intron. We propose a model of plant gene splicing, where multiple exon of individual gene is in favour of efficient CTS likely involving RZ-1C cooperative interactions with many exons and splicing factors. Our work uncovers the robustness of plant CTS and highlighted the role of RZ-1C in this process.
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Project description:Efficient targeted control of exon splicing is a major goal of functional genomic and therapeutic applications. Guide RNA-directed, deactivated (d)Cas CRISPR enzymes fused to splicing effectors represent a promising strategy due to the flexibility and presumed specificity of these systems. However, efficient, selective, and generalizable activation of targeted endogenous exons using this approach has not been reported. Here, we identify dCasRx-RBM25 as a potent activator of exons by screening over 300 dCasRx splicing factor fusions tethered to splicing reporters. dCasRx-RBM25 also strongly activates splicing of endogenous alternative exons, when recruited to downstream intron sequences using single guide RNAs. In transcriptome wide analyses we observe a high degree of specificity of dCasRx-RBM25 for endogenous exon targeting. We further leverage the guide array-processing activity of dCasRx to simultaneously target multiple endogenous exons for activation and repression by dCasRx-RBM25. Our results pave the way for versatile exon-resolution functional assays and splicing-directed therapeutic applications.
2024-09-19 | GSE243979 | GEO
Project description:Universal alternative splicing of noncoding exons
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Project description:The human transcriptome is so large, diverse and dynamic that, even after a decade of investigation by RNA sequencing (RNA-Seq), we are yet to resolve its true dimensions. RNA-Seq suffers from an expression-dependent bias that impedes discovery of low-abundance transcripts and has prevented a complete census of gene expression. Here we performed targeted single-molecule and short-read RNA-Seq to survey the transcriptional landscape of a single human chromosome (Hsa21) at unprecedented resolution. Our analysis reaches the lower limits of the transcriptome and identifies a fundamental distinction between the architecture of protein-coding and noncoding gene content. Unlike their coding counterparts, noncoding exons undergo universal alternative splicing to produce a seemingly limitless variety of isoforms. Targeted RNA-Seq analysis of syntenic regions of the mouse genome shows that few noncoding exons are shared between human and mouse. Despite this divergence, human alternative splicing profiles are recapitulated on Hsa21 in mouse cells, indicative of regulation by a local splicing code that is more strongly conserved than the noncoding isoforms themselves. We propose that noncoding exons are functionally modular, with combinatorial alternative splicing generating an enormous repertoire of potential regulatory RNAs and a rich transcriptional reservoir for gene evolution.
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Project description:ost characterized tumor antigens are ‘genomic’, i.e. encoded by canonical, non-canonical or somatically mutated genomic sequences. We investigate here the presentation and immunogenicity of tumor antigens derived from non-canonical mRNA splicing events between coding exons and transposable elements (TEs). Comparing non-small cell lung cancer (NSCLC), an immunogenic tumor type, and diverse non-tumor tissues, we identify several thousand splicing junctions between exons and diverse TE classes. A subset of these junctions is both tumor-specific and shared across patients. HLA-I peptidomic identifies peptides encoded by tumor-specific junctions in primary NSCLC samples and lung tumor cell lines. Recurrent junction-encoded peptides are immunogenic in vitro and CD8+ T cells specific for junction-encoded epitopes are present in tumors and tumor-draining lymph nodes from NSCLC patients. We conclude that non-canonical splicing junctions between exons and TEs represent a source of recurrent, immunogenic tumor-specific antigens in NSCLC cancer patients.
2023-02-27 | PXD034820 | Pride
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.
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Project description:In eukaryotes, a dynamic ribonucleic protein machine known as the spliceosome catalyzes the removal of introns from pre-messenger RNA (pre-mRNA). Recent studies show the process of RNA-synthesis and RNA-processing to be spatio-temporally coordinated, indicating that RNA splicing takes place in the context of chromatin. H2A.Z is a highly conserved histone variant of the canonical histone H2A. In S. cerevisiae, H2A.Z is deposited into chromatin by the SWR1-complex, is found near the 5’ ends of protein-coding genes, and has been implicated in transcription regulation. Here we show that splicing of intron-containing genes in cells lacking H2A.Z is impaired, particularly under suboptimal splicing conditions. Cells lacking H2A.Z are especially dependent on a functional U2 snRNP, as H2A.Z shows extensive genetic interactions with U2 snRNP associated proteins, and RNA-seq reveals introns with non-consensus branch points are particularly sensitive to H2A.Z loss. Consistently, H2A.Z promotes efficient spliceosomal rearrangements involving the U2 snRNP, as H2A.Z loss results in persistent U2 snRNP association and decreased recruitment of downstream snRNPs to nascent RNA. H2A.Z impairs transcription elongation, suggesting that spliceosome rearrangements are tied to H2A.Z’s role in elongation. Depletion of disassembly factor Prp43 suppresses H2A.Z-mediated splice defects, indicating that, in the absence of H2A.Z, stalled spliceosomes are disassembled and unspliced RNAs are released. Together these data demonstrate that H2A.Z is required for efficient pre-mRNA splicing and indicate a role for H2A.Z in coordinating the kinetics of transcription elongation and splicing.
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Project description:In Arabidopsis, RNA Polymerase II (Pol II) often pauses within a few hundred base pairs downstream of the polyadenylation site, reflecting efficient transcriptional termination, but how such pause is regulated remains largely elusive. Here, we studied Pol II dynamics at gene 3’ end by combining comprehensive experiments with mathematical modelling. We generated high-resolution Ser2P Pol II positioning data specifically enriched at gene 3’ end and defined a 3’ end pause index (3’PI). The position but not the extent of the 3’ end pause correlates with termination window size. The 3’PI is not decreased but even mildly increased in the termination deficient mutant xrn3, indicating 3’ end pause is a regulatory step early during the termination and before XRN3 mediated RNA decay that releases the Pol II. Unexpectedly, 3’PI is closely associated with gene exon numbers and co-transcriptional splicing efficiency. Multiple exons genes often display stronger 3’ end pauses and more efficient on-chromatin splicing than genes with few exons. Chemical inhibition of splicing strongly reduces the 3’PI and disrupts its correlation with exon numbers but does not globally impact 3’ end readthrough levels. These results were further confirmed by fitting Pol II positioning data with a mathematical model, which enables the estimation of parameters that define Pol II dynamics. Our work highlights that the number of exons via co-transcriptional splicing is a major determinant of Pol II pausing levels at gene 3’ end in plants.
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Project description:Co-transcriptional splicing of introns is a defining feature of eukaryotic gene expression. We show that the mammalian spliceosome specifically associates with the S5P CTD isoform of RNA polymerase II (Pol II) as it elongates across spliced exons of protein coding genes, both in human Hela and murine lymphoid cell lines. Immuno-precipitation of MNase digested chromatin with phospho CTD specific antibodies reveals that components of the active spliceosome (both snRNA and proteins) form a specific complex with S5P CTD Pol II. Furthermore a dominant splicing intermediate formed by cleavage at intron 5’ss results in the tethering of upstream exons to this complex at all spliced exons. These are invariably connected to upstream spliced constitutive and less frequently to alternative exons. Finally S5P CTD Pol II accumulates over spliced exons but not adjacent introns. We propose that mammalian splicing employs a rapid, co-transcriptional splicing mechanism based on CTD phosphorylation transitions.
2018-10-05 | PXD008197 | Pride