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Cryptic splicing mediates genetic and therapeutic perturbation of human gene expression levels [naRNA_LCLs]

ABSTRACT: Alternative splicing (AS) in human genes is widely viewed as a mechanism for enhancing proteomic diversity. AS can also impact gene expression levels without increasing protein diversity by producing “unproductive” transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD). However, the relative importance of this regulatory mechanism remains underexplored. To better understand the impact of AS-NMD relative to other regulatory mechanisms, we analyzed population-scale genomic data across eight molecular assays, covering various stages from transcription to cytoplasmic decay. We report threefold more unproductive splicing compared to prior estimates using steady-state RNA. This unproductive splicing compounds across multi-intronic genes, resulting in 15% of all transcript molecules from protein-coding genes being unproductive. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are as often associated with NMD-induced expression level differences as with differences in protein isoform usage. Employing the splice-switching drug risdiplam to manipulate AS at hundreds of genes, we find that ~3/4 of drug-induced isoforms are targeted by NMD, suggesting that most aberrant splicing influences expression levels. Our findings suggest much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.

ORGANISM(S): Homo sapiens

PROVIDER: GSE252005 | GEO | 2024/05/10

REPOSITORIES: GEO

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Cryptic splicing mediates genetic and therapeutic perturbation of human gene expression levels [Cut&Tag]

Project description:Alternative splicing (AS) in human genes is widely viewed as a mechanism for enhancing proteomic diversity. AS can also impact gene expression levels without increasing protein diversity by producing “unproductive” transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD). However, the relative importance of this regulatory mechanism remains underexplored. To better understand the impact of AS-NMD relative to other regulatory mechanisms, we analyzed population-scale genomic data across eight molecular assays, covering various stages from transcription to cytoplasmic decay. We report threefold more unproductive splicing compared to prior estimates using steady-state RNA. This unproductive splicing compounds across multi-intronic genes, resulting in 15% of all transcript molecules from protein-coding genes being unproductive. Leveraging genetic variation across cell lines, we find that GWAS trait-associated loci explained by AS are as often associated with NMD-induced expression level differences as with differences in protein isoform usage. Employing the splice-switching drug risdiplam to manipulate AS at hundreds of genes, we find that ~3/4 of drug-induced isoforms are targeted by NMD, suggesting that most aberrant splicing influences expression levels. Our findings suggest much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.

2024-05-10 | GSE252003 | GEO

Nonsense-mediated decay of alternative precursor mRNA splicing variants is a major determinant of the eukaryotic steady state transcriptome

Project description:Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance pathway that recognizes aberrant transcripts arising from mutations and transcriptional mistakes. Moreover, differential transcript processing such as alternative precursor mRNA splicing (AS) can generate NMD substrates, however, the extent of coupled AS and NMD remained unclear. To investigate NMD targeting of AS variants, we performed transcriptome-wide splicing studies using Arabidopsis thaliana single and double mutants in the NMD factor homologues UPF1 and UPF3, as well as samples treated with the translation inhibitor cycloheximide (CHX). Our analyses revealed that at least 17.5% of all multi-exon, protein-coding genes produce splicing variants of all major types that are targeted by NMD, with a significant fraction originating from the splicing of cryptic introns. Furthermore, accumulation of many intron-retained mRNAs in the mutants, but not in response to CHX suggests the action of distinct routes of NMD with variable impact of translation. Importantly, 92.3% of the NMD-responsive mRNAs exhibit classical NMD-eliciting features, supporting their authenticity as direct targets. NMD-dependent AS variants are linked to diverse biological functions, including the poison exon-mediated regulation of signaling and posttranslational protein modification components. In addition to mRNAs, numerous non-coding RNAs as well as newly identified transcripts derived from intergenic regions were shown to be NMD responsive. In summary, our comprehensive analysis of AS-coupled NMD provides strong evidence for a major function of this pathway in shaping the transcriptome, having fundamental implications in gene regulation and quality control of transcript processing steps in higher eukaryotes. Comparison of gene expression and alternative splicing patterns in control and nonsense-mediated decay (NMD)-impaired Arabidopsis seedlings

2013-10-01 | E-GEOD-41432 | biostudies-arrayexpress

Alternative splicing coupled mRNA decay shapes the temperature-dependent transcriptome

Project description:Mammalian body temperature oscillates with the time of the day and is altered in diverse pathological conditions. We recently identified a body temperature-sensitive thermometer-like kinase, which alters SR protein phosphorylation and thereby globally controls alternative splicing (AS). AS can generate unproductive variants which are recognized and degraded by diverse mRNA decay pathways – including nonsense-mediated decay (NMD). Here we show extensive coupling of body temperature-controlled AS to mRNA decay, leading to global control of temperature-dependent gene expression (GE). Temperature-controlled, decay-inducing splicing events are evolutionarily conserved and pervasively found within RNA-binding proteins, including most SR proteins. AS-coupled poison exon inclusion is essential for rhythmic GE of SR proteins and has a global role in establishing temperature-dependent rhythmic GE profiles, both, in mammals under circadian body temperature cycles and in plants in response to ambient temperature changes. Together, these data identify body temperature-driven AS coupled mRNA decay as an evolutionary ancient, core clock-independent mechanism to generate rhythmic GE.

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Widespread Use of Non-Productive Alternative Splice Sites in Saccharomyces cerevisiae

Project description:Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many Saccharomyces cerevisiae intron-containing genes exhibit usage of alternative splice sites, but most transcripts generated by splicing from these sites are non-functional because they introduce premature termination codons leading to transcript degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3M-bM-^@M-^Y splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3M-bM-^@M-^Y-splice site. The use of non-productive alternative splice sites can limit the expression of some transcripts and can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results reveal that alternative splicing is frequent in S.cerevisiae but masked by RNA degradation and that the use of alternative splice sites is mostly aimed at controlling transcript levels rather than increasing proteome diversity. mRNA-Seq profiling of 3 mutants in the nonsense-mediated mRNA decay pathway and wildtype yeast

2014-02-21 | E-GEOD-55213 | biostudies-arrayexpress

Deep sequencing of pre-translational mRNPs reveals hidden flux through evolutionarily conserved AS-NMD pathways

Project description:Background: Alternative splicing (AS), which generates multiple mRNA isoforms from single genes, is crucial for the regulation of eukaryotic gene expression. The flux through competing AS pathways cannot be determined by traditional RNA-Seq, however, because different mRNA isoforms can have widely differing decay rates. Indeed, some mRNA isoforms with extremely short half-lives, such as those subject to translation-dependent nonsense-mediated decay (AS-NMD), may be completely overlooked in even the most extensive RNA-Seq analyses. Results: RNA immunoprecipitation in tandem (RIPiT) of exon junction complex (EJC) components allows for purification of post-splicing mRNA-protein particles (mRNPs) not yet subject to translation (pre-translational mRNPs) and, therefore, translation-dependent mRNA decay. Here we compared EJC RIPiT-Seq to whole cell RNA-Seq data from HEK293 cells. Consistent with expectation, the flux through known AS-NMD pathways is substantially higher than that captured by RNA-Seq. Our EJC RIPiT-Seq also definitively demonstrates that the splicing machinery itself has no ability to detect reading frame. We identified thousands of previously unannotated splicing events; while many can be attributed to “splicing noise”, others are evolutionarily-conserved events that produce new AS-NMD isoforms likely involved in maintenance of protein homeostasis. Several of these occur in genes whose overexpression has been linked to poor cancer prognosis. Conclusions: Deep sequencing of RNAs in post-splicing, pre-translational mRNPs provides a means to identify and quantify splicing events without the confounding influence of differential mRNA decay. For many known AS-NMD targets, the NMD-linked AS pathway predominates. EJC RIPiT-Seq also enabled identification of numerous conserved but previously unknown AS-NMD events.

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Widespread Use of Non-Productive Alternative Splice Sites in Saccharomyces cerevisiae

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