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Intron looping is mediated during transcription by U1 snRNP and RNA polymerase II [RIP-seq]

ABSTRACT: In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns, we discovered that the 5 splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. Correlation with splicing outcomes demonstrate that these associations are functional. The interactions between 5 splice sites, U1 snRNP, and elongating RNA polymerase II occurs genome-wide. Our findings reveal that during intron synthesis the upstream 5 splice site remains attached to the transcriptional machinery and is thus brought into proximity of the 3 splice site to enable rapid splicing.

ORGANISM(S): Homo sapiens

PROVIDER: GSE144905 | GEO | 2021/06/16

REPOSITORIES: GEO

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Intron looping is mediated during transcription by U1 snRNP and RNA polymerase II [RNA-Seq]

Project description:In the earliest step of spliceosome assembly, the two splice sites flanking an intron are brought into proximity by U1 snRNP and U2AF. The mechanism that facilitates this intron looping is poorly understood. Using a CRISPR interference-based approach to halt RNA polymerase II transcription in the middle of introns, we discovered that the 5 splice site base pairs with a U1 snRNA that is tethered to RNA polymerase II during intron synthesis. Correlation with splicing outcomes demonstrate that these associations are functional. The interactions between 5 splice sites, U1 snRNP, and elongating RNA polymerase II occurs genome-wide. Our findings reveal that during intron synthesis the upstream 5 splice site remains attached to the transcriptional machinery and is thus brought into proximity of the 3 splice site to enable rapid splicing.

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LUC7 is an U1snRNP component that plays a role in alternative splicing and terminal introns removal in plants

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Neuronal stimulation changes U1 snRNP binding and intronic polyadenylation

Project description:Synaptic activity induces well-known changes in enhancer-promoter driven gene expression but also induces changes in splicing and polyadenylation that are understudied. Here, we investigate the mechanism of expression for alternative polyadenylation isoform Homer1a, an immediate early gene essential to synaptic plasticity. We report that neuronal activation, in neuronal cultures and in adult mouse brain, depletes the splice factor U1 snRNP from Homer1 pre-mRNA and that this causes shifted utilization of a cryptic polyadenylation signal within intron 5 resulting in Homer1a expression. Because U1 snRNP is a ubiquitous splice factor, we tested the generality of activity-driven U1 snRNP depletion as a mechanism for gene expression using RNA immunoprecipitation sequencing. Analysis reveals that neuronal activity changes U1 snRNP binding to ~2000 transcripts and for a subset of transcripts, a reduction in U1 snRNP binding was accompanied by utilization of a cryptic intronic polyadenylation site. This subset is enriched for transcripts encoding synaptic proteins involved in excitability control. Genes demonstrating activity-dependent reduced U1 snRNP binding often encode a binding motif for Sam68, a neuronal alternative polyadenylation factor. Findings reveal that activity-driven changes in intron utilization for transcript termination serves an important role in synaptic plasticity.

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2024-10-28 | GSE275154 | GEO

Cooperative engagement and subsequent selective displacement of SR proteins define the pre-mRNA 3D structural scaffold for early spliceosome assembly

Project description:We recently reported that serine-arginine-rich (SR) protein-mediated pre-mRNA structural remodeling generates a pre-mRNA 3D structural scaffold that is stably recognized by the early spliceosomal components. However, the intermediate steps between the free pre-mRNA and the assembled early spliceosome are not yet characterized. By probing the early spliceosomal complexes in vitro and RNA-protein interactions in vivo, we show that the SR proteins bind the pre-mRNAs cooperatively generating a substrate that recruits U1 snRNP and U2AF65 in a splice signal-independent manner. Excess U1 snRNP selectively displaces some of the SR protein molecules from the pre-mRNA generating the substrate for splice signal-specific, sequential recognition by U1 snRNP, U2AF65, and U2AF35. Our work thus identifies a novel function of U1 snRNP in mammalian splicing substrate definition, explains the need for excess U1 snRNP compared to other U snRNPs in vivo, demonstrates how excess SR proteins could inhibit splicing, and provides a conceptual basis to examine if this mechanism of splicing substrate definition is employed by other splicing regulatory proteins.

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Project description:Alternative splicing of pre-mRNAs increases the potential for regulation and complexity of gene expression. The exon junction complex (EJC) and its associated splicing factor RNPS1 were recently shown to suppress mis-splicing resulting from the usage of cryptic and reconstituted 5’ and 3’ splice sites in the vicinity of the EJC. Here, we aimed to further investigate the mechanisms underlying splicing regulation by RNPS1. A transcriptome-wide analysis identified hundreds of splice events affected by the knockdown (KD) of RNPS1 in HeLa cells. These included alternative splice site usage as well as intron retention, exon skipping and inclusion. However, only a fraction of these RNPS1-dependent splice events was fully or partially rescued by the expression of the RNPS1 RRM. These results indicated that another domain of RNPS1 is involved in the regulation of the majority of splicing events. Deletion experiments revealed that the N-terminus and S-domain, and in particular the C-terminus of RNPS1 strongly regulate these events. Several splicing factors, including SR proteins and U1 snRNP components, were strongly reduced in the interactome of RNPS1 lacking the C terminus. We conclude that RNPS1 interacts with many splicing factors to direct the assembly of EJC-dependent and-independent splicing complexes.

2022-05-31 | PXD027251 | Pride

Discovery of pre-mRNA structural scaffold as a contributor to mammalian splicing code [In Vivo]

Project description:The specific recognition of splice signals at or near the exon-intron junctions is not explained by their weak conservation across the mammalian transcriptome and postulated to require a multitude of features embedded in the pre-mRNA strand. We explored the possibility of three-dimensional structural scaffold of a pre-mRNA guiding early spliceosomal components to the splice signal sequences. We find that mutation in non-cognate splice signal sequences of a model pre-mRNA substrate could impede recruitment of early spliceosomal components due to disruption of global structure of the pre-mRNA. We also find distribution of pre-mRNA segments potentially interacting with early spliceosomal component U1 snRNP across the intron, spatial proximity of 5′ and 3′ splice sites within the pre-mRNA scaffold, and an interplay between the structural scaffold and splicing regulatory elements in recruiting early spliceosomal components. These results suggest that early spliceosomal components could recognize a three-dimensional structural scaffold beyond the short splice signal sequences and that in our model pre-mRNA, this scaffold is formed across the intron involving the major splice signals. This work provides a conceptual base to extend our understanding of prevalence, distribution, and splicing regulatory potential of recognizable three-dimensional structural scaffolds across the mammalian transcriptome.

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