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Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans

ABSTRACT: Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing.

ORGANISM(S): Schizosaccharomyces pombe

PROVIDER: GSE48594 | GEO | 2013/07/09

SECONDARY ACCESSION(S): PRJNA210769

REPOSITORIES: GEO

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Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans

Project description:Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing. Total RNA from ∆dbr1 S. pombe grown under 41 different conditions, pooled, then run under two-dimensional gel electrophoresis to separate linear from circular RNA. Circular RNA was excised and prepared as a single-end barcoded Illumina sequencing library.

2013-07-09 | E-GEOD-48594 | biostudies-arrayexpress

Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans.

Project description:Alternative splicing is a potent regulator of gene expression that vastly increases proteomic diversity in multicellular eukaryotes and is associated with organismal complexity. Although alternative splicing is widespread in vertebrates, little is known about the evolutionary origins of this process, in part because of the absence of phylogenetically conserved events that cross major eukaryotic clades. Here we describe a lariat-sequencing approach, which offers high sensitivity for detecting splicing events, and its application to the unicellular fungus, Schizosaccharomyces pombe, an organism that shares many of the hallmarks of alternative splicing in mammalian systems but for which no previous examples of exon-skipping had been demonstrated. Over 200 previously unannotated splicing events were identified, including examples of regulated alternative splicing. Remarkably, an evolutionary analysis of four of the exons identified here as subject to skipping in S. pombe reveals high sequence conservation and perfect length conservation with their homologs in scores of plants, animals, and fungi. Moreover, alternative splicing of two of these exons have been documented in multiple vertebrate organisms, making these the first demonstrations of identical alternative-splicing patterns in species that are separated by over 1 billion y of evolution.

| S-EPMC3732940 | biostudies-literature

A comprehensive map of intron branchpoints and lariat RNAs in plants

Project description:Lariat is formed by excised intron, in which the 5' splice site joints with the branchpoint (BP) during splicing. Although lariat RNAs are usually degraded by DBR1 (RNA debranching enzyme 1), recent findings in animals showed that many lariat RNAs were widely detected under physiological conditions. In contrast, the features of BPs and to what extent lariat RNAs accumulate naturally are largely unexplored in plants. Here, we analyzed 948 RNA sequencing datasets to document plant BPs and lariat RNAs on a genome-wide scale. In total, we identified 13872, 5199, 29582, and 13478 BPs in Arabidopsis, tomato, rice, and maize, respectively. Features of plant BPs are highly similar to those in yeast and human, in that BPs are adenine-preferred and flanked by uracil-enriched sequences. Intriguingly, ~20% of introns harbor multiple BPs, and BP usage is tissue-specific. Furthermore, 10580 lariat RNAs accumulate in wild type Arabidopsis plants, and most of these lariat RNAs originate from longer or retroelement-depleted introns, and the expression of these lariat RNAs significantly correlated with the incidence of back-splicing of parent exons. Collectively, our results provide the first comprehensive map of intron BPs and lariat RNAs in plants, and uncover a novel link between lariat turnover and splicing.

2019-04-17 | GSE117416 | GEO

Lariat mapping of DBR1 KO and DBR1 KO+DBR1 addback cells

Project description:We constructed a DBR1 knockout cell line (C22) using CRISPR in HEK293T cells. Through mapping of lariat reads, lariat levels in the DBR1 - samples are shown to increase dramatically (~20x) relative to wild type cells. Over 60% of this increase in lariat levels is abrogated upon rescue of DBR1 - cells with a DBR1 expression vector

2022-01-29 | GSE195469 | GEO

Profiling lariat intermediates reveals genetic determinants of early and late co-transcriptional splicing

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.

2022-11-26 | GSE204834 | GEO

Lariat mapping of wild type and DBR1 KO cells after knockdown of AQR

Project description:Using wild type HEK293T cells and our DBR1 CRISPR knockout cell line (C22), we investigated the effect of AQR knockdown on cellular lariat levels. Samples were taken after transfection with either one of two AQR-targeting siRNAs or a non-targeting control siRNA. After sequencing the samples, lariat mapping of the reads revealed that AQR knockdown increased the lariat recovery rate by ~50% relative to the level observed in the negative control samples.

2022-01-31 | GSE195668 | GEO

RNAseq and Lariat sequencing of different S. pombe growth conditions

Project description:We performed lariat sequencing to profile the diversity of spliced RNA lariats in S. pombe identify annotated and alternate introns. Three different growth conditions were used to grow S. pombe wt and S. pombe Δdbr1. Lariat sequencing of the Δdbr1 strains and RNAseq of the wt and Δdbr1 strains were done to profile intron lariats and exon-exon junctions in RNA transcripts.

2015-09-03 | E-GEOD-68345 | biostudies-arrayexpress

The Debranching Enzyme Dbr1 Regulates Lariat Turnover and Intron Splicing

Project description:The majority of genic transcription is intronic. Introns are removed by splicing as branched lariat RNAs which require rapid recycling. The branch site is recognized during splicing catalysis and later debranched by Dbr1 in the rate-limiting step of lariat turnover. Through generation of the first viable DBR1 knockout cell line, we find the predominantly nuclear Dbr1 enzyme to encode the sole debranching activity in human cells. Dbr1 preferentially debranches substrates that contain canonical U2 binding motifs, suggesting that branchsites discovered through sequencing do not necessarily represent those favored by the spliceosome. We find that Dbr1 also exhibits specificity for particular 5' splice site sequences. We identify Dbr1 interactors through co-immunoprecipitation mass spectroscopy. We present a mechanistic model for Dbr1 recruitment to the branchpoint through the intron-binding protein AQR. In addition to a 20-fold increase in lariats, Dbr1 depletion increases exon skipping. Using ADAR fusions to timestamp lariats, we demonstrate a defect in spliceosome recycling. In the absence of Dbr1, spliceosomal components remain associated with the lariat for a longer period of time. As splicing is co-transcriptional, slower recycling increases the likelihood that downstream exons will be available for exon skipping.

2023-06-26 | MSV000092263 | MassIVE

RNAseq and Lariat sequencing of different S. pombe growth conditions

Project description:We performed lariat sequencing to profile the diversity of spliced RNA lariats in S. pombe identify annotated and alternate introns.

2015-09-03 | GSE68345 | GEO

Schizosaccharomyces pombe

Project description:Lariat sequencing in a unicellular yeast identifies regulated alternative splicing of exons that are evolutionarily conserved with humans

| PRJNA210769 | ENA

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