Biology subjects

Valcarcel, J.

Publications and source records attributed to Valcarcel, J..

9 recordsLinked to original sources

bioRxiv2026

RERE and the Mediator complex cooperate with EWSR1::FLI1 in the reprogramming of Translation and Alternative Splicing, the latter being a therapeutically targetable vulnerability in Ewing sarcoma

Ewing Sarcoma (ES) is an aggressive neoplasm arising in bones and soft tissues driven by the oncogenic fusion EWSR1::FLI1. Through epigenetic deregulation, EWSR1::FLI1 generates de novo super-enhancers that control the expression of key genes for tumor cell maintenance. By an integrative in silico analysis, we identified the subunit of the Mediator complex MED13L and RERE, a member of the atrophin family of arginine-glutamic acid dipeptide repeat-containing proteins, as genes regulated by EWSR1::FLI1-bound super-enhancers. We confirmed that EWSR1::FLI1 regulates MED13L and RERE expression in ES cell lines and showed that these proteins are highly expressed in Ewing primary tumors. Besides the well-established role of the Mediator complex in transcriptional regulation given its association with the RNA polymerase II, in ES cells the DNA binding sites of MED13L overlap with those of RERE and EWSR1::FLI1 in genes that control protein translation and alternative splicing (AS). Accordingly, the expression of various spliceosome components is co-regulated by MED13L, RERE and the oncogene, leading to AS in ES cells. We identified RBM39, a splicing factor downregulated after MED13L and RERE depletion, as a direct transcriptional target of EWSR1::FLI1. Consistently, in vitro viability experiments using indisulam, which induces selective DCAF15-dependent proteosome degradation of RBM39, demonstrate ES cells highly and specifically sensitive to RBM39 inhibition. In vivo experiments with mice xenografted with ES cells show complete tumor regression with indisulam, highlighting the potential of this approach as a novel and promising therapeutic strategy for Ewing sarcoma. STATEMENT OF SIGNIFICANCEEwing sarcoma (ES) is characterized by FET::ETS oncoproteins that act as pioneer transcription factors. Here, we identified two genes controlled by EWSR1::FLI1-bound super-enhancers, MED13L and RERE, and characterized the mechanism by which these proteins cooperate with the oncogene to regulate RNA metabolism and ribosomal processes in ES cells. These findings have led to the identification of the splicing factor RBM39 as a vulnerability in ES, as supported by the extraordinary sensitivity of these tumors to monotherapy with RBM39 degrader indisulam.

cancer biology↗

bioRxiv2026

OpenSplice: the impact of half a million mutations on the alternative splicing of 600 human exons

Alternative splicing of mRNA precursors is an important step in gene regulation and a major mechanism by which genetic variants cause human disease. However, changes in splicing have only been quantified for a tiny fraction of possible variants in the human genome, limiting our ability to interpret clinical variants, evaluate and develop machine learning models, and understand the splicing regulatory code. Here, to address this data gap, we present OpenSplice, a well-calibrated experimental dataset that quantifies the impact of >590,000 variants on the splicing of >600 human alternatively spliced exons. OpenSplice increases the number of exons with site saturation mutagenesis data [~]28-fold, quantifying the impact of all possible exonic and proximal intronic single nucleotide (nt) substitutions, as well as all 1, 3, 6 and 21nt deletions. Hundreds of thousands of variants affect splicing and we use the data to evaluate machine learning models, to interpret clinical variants, and to map splicing regulatory architectures. Exons and introns exhibit a broad spectrum of regulatory landscapes, including configurations dominated by enhancers or silencers, checkerboard-like patterns with interleaved enhancers and silencers, and sparse architectures with minimal regulatory content. Silencers are particularly important for determining inclusion levels. OpenSplice provides a complete atlas of variants that impact splicing, a rich testing and training dataset for machine learning models, and a comprehensive map of regulatory elements for mechanistic studies.

genomics↗

bioRxiv2025

A splice-switching antisense oligonucleotide approach for pediatric genetic epilepsies

Summary ParagraphVariants in ion channel genes are common causes of pediatric epilepsy, often leading to intractable seizures, developmental delay and other comorbidities, which increases risk of death. Pathogenic variants in the SCN8A gene, which encodes a voltage-gated sodium channel critical for action potential generation in the brain, account for [~]1% of genetic epilepsies. The voltage sensor in SCN8A domain 1 is encoded by one of two developmentally-regulated mutually exclusive alternative exons, 5N and 5A. We observe that variants in these exons are more likely to cause infantile spasms, a severe seizure type, than variants elsewhere in SCN8A, and that some pathogenic variants affect exon 5 splicing, impacting patient phenotype. Molecular and evolutionary analyses implicate the exon sequences of these and other voltage-gated ion channel alternative exons in splicing regulation. We identified antisense oligonucleotides (ASOs) that shift splicing of SCN8A exon 5N to 5A or vice versa. These ASOs normalize neuronal activity in patient-derived iPSC neurons, and reduce seizures and motor impairment and extend lifespan in a new exon 5N mutant mouse model. Our results demonstrate that splice-switching ASOs can effectively reduce the expression of pathogenic isoforms and rescue both seizure and non-seizure phenotypes. Similar approaches should be applicable to pediatric genetic epilepsies caused by mutations in other ion channel alternative exons.

neuroscience↗

bioRxiv2024

Deep indel mutagenesis reveals the regulatory and modulatory architecture of alternative exon splicing

Altered splicing is a frequent mechanism by which genetic variants cause disease and antisense oligonucleotides (AONs) that target pre-mRNA splicing have been approved as therapeutics for multiple pathologies including patient-customized treatments for rare diseases. However, the regulatory architecture of human exons remains poorly understood and AON discovery is currently slow and expensive, limiting the wider adoption of the approach. Here we show that that systematic deletion scans -which can be made experimentally at very low cost - provide an efficient strategy to chart the regulatory landscape of human exons and to rapidly identify effective splicing-modulating oligonucleotides in a fully quantitative manner. Our results suggest a mechanism for the evolutionary origins of unusually short microexons and the repression of transmembrane domain-encoding exons, and reveal a checkerboard architecture of sequential enhancers and silencers in a model alternative exon. Accurate prediction of the effects of deletions using deep learning provides a resource, DANGO, that maps the splicing regulatory landscape of all human exons and predicts effective splicing-altering antisense oligonucleotides genome-wide.

genetics↗

bioRxiv2022

Structural basis for specific RNA recognition by the alternative splicing factor RBM5

The RNA-binding motif protein RBM5 belongs to a family of multi-domain RNA binding proteins that are implicated in cancer and regulate alternative splicing of genes important for apoptosis and cell proliferation and have been implicated in cancer. RBM5 harbors structural modules for RNA recognition, such as RRM domains and a Zn finger, and protein-protein interactions such as an OCRE domain. Here, we characterize binding of the RBM5 RRM1-ZnF1-RRM2 domains to cis-regulatory RNA elements. A structure of the RRM1-ZnF1 region in complex with RNA shows how the tandem domains cooperate to sandwich target RNA and specifically recognize a GG dinucleotide in a non-canonical fashion. While the RRM1-ZnF1 domains act as a single structural module, RRM2 is connected by a flexible linker and tumbles independently. However, all three domains participate in RNA binding and adopt a closed architecture upon RNA binding. Our data highlight how cooperativity and conformational modularity of multiple RNA binding domains enable the recognition of distinct RNA motifs, thereby contributing to the regulation of alternative splicing. Remarkably, we observe surprising differences in coupling of the RNA binding domains between the closely related homologs RBM5 and RBM10. HighlightsO_LIMultiple RNA binding domains enable differential recognition of distinct RNA motifs to regulate alternative splicing C_LIO_LIThe RRM1-ZnF1 domains of RBM5 mediate specific recognition of cis regulatory RNA motifs to modulate alternative splicing C_LIO_LIRRM1-ZnF1 sandwich the target RNA for non-canonical recognition of a GG dinucleotide C_LIO_LIRRM1-ZnF1-RRM2 bind cooperatively to contiguous cis-regulatory motifs in caspase-2 and NUMB pre-mRNAs C_LI

biochemistry↗

bioRxiv2022

Control of pancreatic islet function and glucose homeostasis by a novel microexon program misregulated in type 2 diabetes

Pancreatic islets control glucose homeostasis by the balanced secretion of insulin and other hormones, and their abnormal function causes diabetes or hypoglycemia. Here, we uncover a conserved program of alternative microexons included in mRNAs of islet cells, particularly in genes involved in vesicle transport and exocytosis. Islet microexons (IsletMICs) are regulated by the RNA binding protein SRRM3 and represent a subset of the larger neural program that are particularly sensitive to the levels of this regulator. Both SRRM3 and IsletMICs are induced by elevated glucose levels, and depletion of SRRM3 in beta cell lines and mouse islets, or repression of particular IsletMICs using antisense oligonucleotides, leads to inappropriate insulin secretion. Consistently, SRRM3 mutant mice display defects in islet cell identity and function, leading to hyperinsulinemic hypoglycemia. Importantly, human genetic variants that influence SRRM3 expression and IsletMIC inclusion in islets are associated with fasting glucose variation and type 2 diabetes risk.

molecular biology↗

bioRxiv2020

Aurora-A phosphorylates splicing factors and regulates alternative splicing

Aurora-A kinase is well known to regulate progression through mitosis. However, the kinase also performs additional functions that could explain the failure of its inhibitors to be effective in cancer treatments. To identify these functions, we applied a proteomics approach to search for interactors of Aurora-A. We found a large number of proteins involved in pre-mRNA splicing, strongly suggesting an important role for Aurora-A in this biological process. Consistently, we first report the subcellular localization of Aurora-A in nuclear speckles, the storehouse of splicing proteins. We also demonstrate direct interaction of Aurora-A with RRM domain-containing splicing factors such as hnRNP and SR proteins and their phosphorylation in vitro. Further, RNA-sequencing analysis following pharmacological inhibition of Aurora-A resulted in alternative splicing changes corresponding to 505 genes, including genes with functions regulated by Aurora-A kinase. Finally, we report enrichment of RNA motifs within the alternatively spliced regions affected by Aurora-A kinase inhibition which are bound by Aurora-A interacting splicing factors, suggesting that Aurora-A regulates alternative splicing by modulating the activity of these interacting splicing factors. Overall our work identified Aurora-A as a novel splicing kinase and for the first time, describes a broad role of Aurora-A in regulating alternative splicing.

molecular biology↗

bioRxiv2020

Dynamics of alternative splicing during somatic cell reprogramming reveals functions for RNA-binding proteins CPSF3, hnRNP UL1 and TIA1

In contrast to the extensively studied rewiring of epigenetic and transcriptional programs required for cell reprogramming, the dynamics of post-transcriptional changes and their associated regulatory mechanisms remain poorly understood. Here we have studied the dynamics of alternative splicing (AS) changes occurring during efficient reprogramming of mouse B cells into induced pluripotent stem (iPS) cells. These changes, generally uncoupled from transcriptional regulation, significantly overlapped with splicing programs reported during reprogramming of mouse embryonic fibroblasts (MEFs). Correlation between gene expression of potential regulators and specific clusters of AS changes enabled the identification and subsequent validation of CPSF3 and hnRNP UL1 as facilitators, and TIA1 as repressor of MEFs reprogramming. These RNA-binding proteins control partially overlapping programs of splicing regulation affecting genes involved in developmental and morphogenetic processes. Our results reveal common programs of splicing regulation during reprogramming of different cell types and identify three novel regulators of this process.

molecular biology↗

bioRxiv2020

Mutations primarily alter the inclusion of alternatively spliced exons

Genetic analyses and systematic mutagenesis have revealed that synonymous, non-synonymous and intronic mutations frequently alter the inclusion levels of alternatively spliced exons, consistent with the concept that altered splicing might be a common mechanism by which mutations cause disease. However, most exons expressed in any cell are highly-included in mature mRNAs. Here, by performing deep mutagenesis of highly-included exons and by analysing the association between genome sequence variation and exon inclusion across the transcriptome, we report that mutations only very rarely alter the inclusion of highly-included exons. This is true for both exonic and intronic mutations as well as for perturbations in trans. Therefore, mutations that affect splicing are not evenly distributed across primary transcripts but are focussed in and around alternatively spliced exons with intermediate inclusion levels. These results provide a resource for prioritising synonymous and other variants as disease-causing mutations.

genomics↗