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An essential role for the ultra-conserved poison exon of Tra2b in fertility in mice (iCLIP-seq)

ABSTRACT: Alternative splicing-induced inclusion of poison exons containing in-frame stop codons is a mechanism that can be used to attenuate gene expression. Poison exon have been implicated in cancer, but how they operate within the context of normal development and physiology is poorly understood. Several splicing regulator genes, including Tra2b, contain ultra-conserved poison exons that function within regulatory loops to fine-tune their activity. To investigate the physiological role of poison exons in vivo, we created mice lacking either Tra2b or its poison exon, specifically during spermatogenesis to reveal both are essential for male fertility. The mouse Tra2b gene is essential for mitotic proliferation of germ cells, whereas, in contrast, the Tra2b poison exon is critically required during meiosis and not needed by mitotically proliferating cell populations within the germline. Poison exon deletion causes infertility, with a block in male meiotic prophase where Tra2β protein expression levels normally increase. Deletion of the Tra2b poison exon changes expression patterns of genes important for meiosis and splicing patterns of Tra2β target exons, suggesting Tra2b poison exon splicing prevents meiotic cells accumulating toxic levels of Tra2b expression. Our data provide a new physiological explanation for Tra2b poison exon ultra-conservation and indicate the importance of evaluating poison exon function within a physiological context.

ORGANISM(S): Mus musculus

PROVIDER: GSE235083 | GEO | 2024/12/19

REPOSITORIES: GEO

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Similar Datasets

An essential role for the ultra-conserved poison exon of Tra2b in fertility in mice (RNA-seq)

Project description:Alternative splicing-induced inclusion of poison exons containing in-frame stop codons is a mechanism that can be used to attenuate gene expression. Poison exon have been implicated in cancer, but how they operate within the context of normal development and physiology is poorly understood. Several splicing regulator genes, including Tra2b, contain ultra-conserved poison exons that function within regulatory loops to fine-tune their activity. To investigate the physiological role of poison exons in vivo, we created mice lacking either Tra2b or its poison exon, specifically during spermatogenesis to reveal both are essential for male fertility. The mouse Tra2b gene is essential for mitotic proliferation of germ cells, whereas, in contrast, the Tra2b poison exon is critically required during meiosis and not needed by mitotically proliferating cell populations within the germline. Poison exon deletion causes infertility, with a block in male meiotic prophase where Tra2β protein expression levels normally increase. Deletion of the Tra2b poison exon changes expression patterns of genes important for meiosis and splicing patterns of Tra2β target exons, suggesting Tra2b poison exon splicing prevents meiotic cells accumulating toxic levels of Tra2b expression. Our data provide a new physiological explanation for Tra2b poison exon ultra-conservation and indicate the importance of evaluating poison exon function within a physiological context.

2024-12-19 | GSE235084 | GEO

Neuronal-specific Deficiency of the Splicing Factor Tra2b Causes p21-mediated Apoptosis in Neurogenic Areas of the Developing Mouse Brain

Project description:Alternative splicing (AS) increases the informational content of the genome and is more prevalent in the brain than in any other tissue. The splicing factor Tra2b (Sfrs10) can modulate splicing inclusion of exons by specifically detecting GAA-rich binding motifs and its absence causes early embryonic lethality in mice. TRA2B has been shown to be involved in splicing processes of Nasp (nuclear autoantigenic sperm protein), MAPT (microtubule associated protein tau) and SMN (survival motor neuron), and is therefore implicated in spermatogenesis and neurological diseases like AlzheimerM-^Rs disease, dementia, ParkinsonM-^Rs disease and spinal muscular atrophy. Here we generated a neuronal-specific Tra2b knock-out mouse that lacks Tra2b expression in neuronal and glial precursor cells by using the Nestin-Cre. Neuronal-specific Tra2b knock-out mice die immediately after birth and show severe abnormalities in cortical development, which are caused by massive apoptotic events in the ventricular layers of the cortex, demonstrating a pivotal role of Tra2b for the developing central nervous system. Using whole brain RNA on exon arrays we identified differentially expressed alternative exons of Tubulin?1 and Shugoshin like2 as in vivo targets of Tra2b. Most interestingly, we found increased expression of the cyclin dependent kinase inhibitor 1a (p21) which we could functionally link to neuronal precursor cells in the affected brain regions. We provide further evidence that the absence of Tra2b causes p21 upregulation and ultimately cell death in NSC34 neuronal-like cells. These findings demonstrate that Tra2b regulates splicing events essential for maintaining neuronal viability during development. Apoptotic events triggered via p21 might not be restricted to the developing brain but could possibly be generalized to the whole organism and explain early embryonic lethality in Tra2b-depleted mice.

2013-12-16 | E-MTAB-2157 | biostudies-arrayexpress

Investigation into human Tra2 protein-dependent splicing in MDA-MB-231 cells using iCLIP and RNA-seq

Project description:Alternative splicing—the production of multiple mRNA isoforms from a single gene—is regulated in part by RNA-binding proteins (RBPs). While the RBPs Tra2α and Tra2β have both been implicated in the regulation of alternative splicing, their relative contribution to this process are not well understood. Here we use iCLIP to identify Tra2β target exons in MDA-MB-231 cells. We find that simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in the splicing pattern of endogenous Tra2β target exons identified by iCLIP. We next use RNA-seq following joint Tra2 protein depletion to comprehensively identify Tra2 protein-dependent exons in MDA-MB-231 cells.

2014-07-12 | GSE59335 | GEO

Ultra-deep sequencing reveals pre-mRNA splicing as a sequence driven high-fidelity process

Project description:Alternative splicing diversifies mRNA transcripts in human cells. While the spliceosome pairs exons with a high degree of accuracy, the rates of rare aberrant and non-canonical pre-mRNA splicing have not been evaluated at the nucleotide level to determine the quantity and identity of these events across splice junctions. Using ultra-deep sequencing the frequency of aberrant and non-canonical splicing events for three splice junctions flanking exon 7 of SMN1 were determined at single nucleotide resolution. After correction for background noise introduced by PCR amplification and sequencing steps, pre-mRNA splicing was shown to maintain a low overall rate of aberrant and non-canonically spliced events. Several previously unannotated splicing events across 3 exon|intron junctions in SMN1 were identified. Mutations within SMN exon 7 were shown to affect splicing fidelity by modulating RNA secondary structures, by altering the binding site of regulatory proteins and by changing the 5’ splice site strength. Mutations also create a truncated SMN1 exon 7 through the introduction of a de novo non-canonical 5’ splice site. The results from the ultra-deep sequencing approach highlight the impressive fidelity of pre-mRNA splicing and demonstrate that the immediate sequence context around splice sites is the main driving force behind non-canonical splice site pairing.

2019-09-24 | GSE137883 | GEO

Conserved role for spliceosomal component PRPF40A in microexon splicing

Project description:Microexons (exons ≤30 nts) are important features of neuronal transcriptomes, but pose mechanistic challenges to the splicing machinery. We previously showed that PRP-40, a component of the U1 spliceosome, is globally required for microexon splicing in C. elegans. Here we show that the homologous PRPF40A is also globally required for microexon splicing in mouse neuroblastoma cells. We find that PRPF40A co-regulates microexons along with SRRM4, a neuron-specific regulator of microexon splicing. The relationship between exon size and dependence on PRPF40A/SRRM4 is distinct, with SRRM4-dependence exhibiting a size threshold (~30 nts) and PRPF40A-dependence exhibiting a graded decrease as exon size increases. Finally, we show that PRPF40A knockdown causes an increase in productive splicing of its spliceosomal binding partner Luc7l by skipping of a small “poison exon.” Similar homeostatic cross-regulation is often observed across paralogous RNA binding proteins. Here we find this concept likewise applies across evolutionarily unrelated but functionally and physically coupled spliceosomal components.

2024-11-05 | GSE267257 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY)

Project description:To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oliogonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTBactivated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons. Target was prepared from 3 biological replicates of PTB/nPTB knockdown and 3 control mock knockdowns from HeLa S3 cell line and hybridized to a custom Affymetrix array containing exon and exon-junction probes for more than 30,000 human genes

2010-09-01 | E-GEOD-23513 | biostudies-arrayexpress

Human introns contain conserved tissue-specific cryptic poison exons

Project description:Eukaryotic cells express a large number of transcripts from a single gene due to alternative splicing. Despite hundreds of thousands of splice isoforms being annotated in databases, it has been reported that the current exon catalogs remain incomplete. At the same time, introns of human protein-coding genes contain a large number of evolutionarily conserved elements with unknown function. Here, we explore the possibility that some of them represent cryptic exons that are expressed in rare conditions. We identified a group of cryptic exons that are similar to the annotated exons in terms of evolutionary conservation and RNA-seq read coverage in the GTEx dataset. Most of them were poison, i.e. generated an NMD isoform upon inclusion, and many showed signs of tissue-specific and cancer-specific expression and regulation. We performed RNA-seq in A549 cell line treated with cycloheximide to inactivate NMD, and confirmed using qPCR that seven of eight exons tested are, indeed, expressed. This study shows that introns of human protein-coding genes contain cryptic poison exons, which reside in conserved intronic regions and remain not fully annotated due to insufficient representation in RNA-seq libraries.

2024-11-20 | GSE270310 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (HJAY)

2010-09-01 | GSE23513 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)

2010-09-01 | GSE23514 | GEO

Position-dependent alternative splicing activity revealed by global profiling of alternative splicing events regulated by PTB (Exon array)

Project description:To gain global insights into the role of the well-known repressive splicing regulator PTB we analyzed the consequences of PTB knockdown in HeLa cells using high-density oliogonucleotide splice-sensitive microarrays. The major class of identified PTB-regulated splicing event was PTB-repressed cassette exons, but there was also a substantial number of PTB-activated splicing events. PTB repressed and activated exons showed a distinct arrangement of motifs with pyrimidine-rich motif enrichment within and upstream of repressed exons, but downstream of activated exons. The N-terminal half of PTB was sufficient to activate splicing when recruited downstream of a PTB-activated exon. Moreover, insertion of an upstream pyrimidine tract was sufficient to convert a PTBactivated to a PTB-repressed exon. Our results demonstrate that PTB, an archetypal splicing repressor, has variable splicing activity that predictably depends upon its binding location with respect to target exons. Target was prepared from 6 biological replicates of PTB/nPTB knockdown and 6 control mock knockdowns from HeLa S3 cell line and hybridized to the Affymetrix Human Exon 1.0 ST Array. Two groups of three replicates each were collected at two different times.

2010-09-01 | E-GEOD-23514 | biostudies-arrayexpress

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