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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE195668 | GEO | 2022/01/31

REPOSITORIES: GEO

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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

Lariat mapping of a DBR1 CRISPR KO cell line

Project description:We used CRISPR in HEK293T cells to create two DBR1 knockout cell lines (C19 and C22). After high-throughput sequencing of total RNA extracted from these cells, we performed lariat read mapping using the method described in "Large-scale mapping of branchpoints in human pre-mRNA transcripts in vivo" (Taggart et al, 2012). We found lariats in the two DBR1- cell lines to be ~20-fold enriched relative to the levels observed in the HEK293T control samples.

2022-02-03 | GSE195586 | GEO

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

Project description:Lariat mapping of DBR1 KO and DBR1 KO+DBR1 addback cells

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

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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.

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Lariat mapping of a DBR1 CRISPR KO cell line

Project description:Lariat mapping of a DBR1 CRISPR KO cell line

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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

Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for correct mRNA splicing and ectodermal differentiation

Project description:The brittle hair syndrome Trichothiodystrophy (TTD) is characterized by variable clinical features, including photosensitivity, ichthyosis, growth retardation, microcephaly, intellectual disability, hypogonadism, and anemia. TTD-associated mutations typically cause unstable mutant proteins involved in various steps of gene-expression, severely reducing steady-state mutant protein levels. However, to date, no such link to instability of gene expression factors for TTD-associated mutations in MPLKIP/TTDN1 has been established. Here, we present seven additional TTD individuals with MPLKIP mutations from five consanguineous families, with a newly identified MPLKIP variant in one family. By mass spectrometry-based interaction proteomics we demonstrate that MPLKIP interacts with core splicing factors and the lariat debranching protein DBR1. MPLKIP-deficient primary fibroblasts have reduced steady-state DBR1 protein levels. Using Human Skin Equivalents (HSEs), we observed impaired keratinocyte differentiation associated with compromised splicing and eventually, an imbalanced proteome affecting skin development and, interestingly, also the immune system. Our data show that MPLKIP, through its DBR1 stabilizing role, is implicated in mRNA splicing, which is of particular importance in highly differentiated tissue.

2023-08-25 | PXD044779 | Pride

Trichothiodystrophy-associated MPLKIP maintains DBR1 levels for correct mRNA splicing and ectodermal differentiation

2023-08-09 | PXD044434 | Pride

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

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