Identification of New Branch Points and Unconventional Introns in Saccharomyces cerevisiae
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA281544 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR1980750_1.fastq.gz | Fastqsanger.gz | |||
| SRR1980750_2.fastq.gz | Fastqsanger.gz | |||
| SRR1980751_1.fastq.gz | Fastqsanger.gz | |||
| SRR1980751_2.fastq.gz | Fastqsanger.gz | |||
| SRR1980742_1.fastq.gz | Fastqsanger.gz |
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Project description:Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BP and 5'SS of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We used RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings reveal BP-based regulation and demonstrate unanticipated complexity of splicing in yeast. 1 Lariat-seq experiment library. 3 barcoded Branch-seq libraries that make up one experiment. 26 RNA-seq samples, 2 biological replicates of each.
2016-08-01 | E-GEOD-68022 | biostudies-arrayexpress
Project description:Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BP and 5'SS of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We used RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings reveal BP-based regulation and demonstrate unanticipated complexity of splicing in yeast.
2016-08-01 | GSE68022 | GEO
Project description:Spliced messages constitute one-fourth of expressed mRNAs in the yeast Saccharomyces cerevisiae, and most mRNAs in metazoans. Splicing requires 5' splice site (5'SS), branch point (BP), and 3' splice site (3'SS) elements, but the role of the BP in splicing control is poorly understood because BP identification remains difficult. We developed a high-throughput method, Branch-seq, to map BPs and 5'SSs of isolated RNA lariats. Applied to S. cerevisiae, Branch-seq detected 76% of expressed, annotated BPs and identified a comparable number of novel BPs. We performed RNA-seq to confirm associated 3'SS locations, identifying some 200 novel splice junctions, including an AT-AC intron. We show that several yeast introns use two or even three different BPs, with effects on 3'SS choice, protein coding potential, or RNA stability, and identify novel introns whose splicing changes during meiosis or in response to stress. Together, these findings show unanticipated complexity of splicing in yeast.
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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: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:Spliceosomal introns are ubiquitous non-coding RNAs typically destined for rapid debranching and degradation. Here, we describe 34 excised Saccharomyces cerevisiae introns that, although rapidly degraded in log-phase growth, accumulate as linear RNAs under either saturated-growth conditions or other stresses that cause prolonged inhibition of TORC1, a key integrator of growth signaling. Introns that become stabilized remain associated with components of the spliceosome and differ from other spliceosomal introns in having a short distance between their lariat branch point and 3´ splice site, which is necessary and sufficient for their stabilization. Deletion of these unusual introns is disadvantageous in saturated conditions and causes aberrantly high growth rates of yeast chronically challenged with the TORC1 inhibitor rapamycin. Reintroduction of native or engineered stable introns suppresses this aberrant rapamycin response. Thus, excised introns function within the TOR growth-signaling network of S. cerevisiae, and more generally, excised spliceosomal introns can have biological functions.
2018-10-29 | GSE121765 | GEO
Project description:Pre-mRNA secondary structures are hypothesized to play widespread roles in regulating RNA processing pathways, but these structures have been difficult to visualize in vivo. Here, we characterize S. cerevisiae pre-mRNA structures through transcriptome-wide dimethyl sulfate (DMS) probing, enriching for low-abundance pre-mRNA through splicing inhibition. These data enable evaluation of structures from phylogenetic and mutational studies as well as identification of new structures across 161 introns. We find widespread formation of “zipper stems” between the 5’ splice site and branch point, “downstream stems” between the branch point and the 3’ splice site, and previously uncharacterized long stems that distinguish pre-mRNA from spliced mRNA. Multi-dimensional chemical mapping reveals that intron structures can form in vitro without the presence of binding partners, and structure ensemble prediction suggests that these structures appear in introns across the Saccharomyces genus. We develop the functional assay VARS-seq to characterize variants of RNA structure in high-throughput and we apply the method on 135 sets of stems across 7 introns, finding that some structured elements can increase spliced mRNA levels despite being distal from canonical splice sites. Unexpectedly, other structures including zipper stems can increase retained intron levels. This transcriptome-wide inference of intron RNA structures suggests new ideas and model systems for understanding how pre-mRNA folding influences gene expression.
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Project description:U12-type introns were originally recognized based on their highly conserved non-consensus AT-AC termini1,2, which are spliced by a separate minor spliceosome3,4. Padgett and Krainer groups later showed that terminal dinucleotides do not differentiate U12-type from U2-type introns, as there are U12-type introns with GT-AG termini and U2-type introns with AT-AC termini5,6. Rather, U12-type introns are recognized by their divergent and highly conserved 5’ splice site (5’ss) and branch point sequences, which both differ from the consensus sequences found in U2-type introns. To date, no functional differences have been ascribed to AT-AC or GT-AG subtypes of U12-type introns, nor have RNAseq analyses of minor spliceosome diseases reported any subtype specificity. Here, we describe a novel protein component of the minor spliceosome, encoded by the CENATAC locus, that is required for accurate splicing of AT-AC but not GT-AG type minor introns. CENATAC was initially identified in a subset of Mosaic Variegated Aneuploidy (MVA) patients with mutations in CENATAC, which lead to chromosome congression defects during mitosis. Earlier large-scale proteomic analyses tentatively classified CENATAC as a spliceosome component and phylogenetic analyses showed co-segregation of CENATAC with minor spliceosome components. Targeted depletion of CENATAC in HeLa cells, followed by RNAseq revealed global retention of AT-AC minor subtype introns with more than 60% showing statistically significant (up to 90%) intron retention (IR). Additionally, U12-type introns with 5’-AT, but divergent 3’-terminal dinucleotides also showed significant IR. We also detected cryptic U2-type splice site activation near affected AT-AC introns. In contrast, about 10% of GT-AG subtype introns responded to CENATAC depletion. Co-IP experiments revealed that CENATAC is not a U11/U12 di-snRNP component as expected for a specificity factor, but rather associates with the U4atac/U6atac.U5 tri-snRNP via interaction with PRPF3/4, suggesting a role for minor tri-snRNP in initial 5’ss recognition. CENATAC also interacts with TXNL4B, a paralog of TXNL4A in the major tri-snRNP. Finally, several genes encoding chromosome congression factors harbor U12 AT-AC-type introns that were highly retained in CENATAC depleted cells, potentially explaining the aneuploidy phenotype observed in MVA patients.
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Project description:Removal of introns by pre-mRNA splicing is a critical and in some cases rate-limiting step in mammalian gene expression. Deep sequencing of mouse embryonic stem cell RNA revealed many specific internal introns that are significantly more abundant than the other introns within poly(A) selected transcripts; we classify these as “detained” introns (DIs). We identified thousands of DIs flanking both constitutive and alternatively spliced exons in human and mouse cell lines. Drug inhibition of Clk SR-protein kinase activity triggered rapid splicing changes in a specific set of DIs, about half of which showed increased splicing and half increased intron detention, altering the transcript pool of over 300 genes. These data suggest a widespread mechanism by which a nuclear detained pool of mostly processed pre-mRNAs can be rapidly mobilized in response to stress or homeostatic autoregulation. v6.5 mouse embryonic stem cells were untreated, treated with the Clk kinase inhibitor KH-CB19, or treated with DMSO as a negative control. Untreated cells were harvested and a single replicate was sequenced using a custom, ligation-based, stranded library preparation protocol. Treated cells were harvested at time 0 and at 2 hours post-treatment, and poly(A)-selected RNA-seq libraries were made from biological duplicates for each treatment/time, barcoded, and sequenced by strand-specific, paired-end sequencing using the Illumina TruSeq kit.
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