ICLIP of Tra2b, Son-GFP and Srrm2-GFP in mESCs
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ABSTRACT:
PROVIDER: PRJEB65872 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:The nucleus of higher eukaryotes is a highly compartmentalized and dynamic organelle consisting of several biological condensates that regulate gene expression at multiple levels. First reported more than 100 years ago by Ramón y Cajal, nuclear speckles (NS) are among the most prominent of such condensates, which were independently rediscovered multiple times and are also known as interchromatin granule clusters (ICGs), splicing speckles, or SC35 domains. Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified. The monoclonal antibody SC35, which was raised against a spliceosomal extract, is a frequently used reagent to mark NS since its debut in 1990. Despite its prolific use, and the consensus regarding its primary target as SRSF2, clear inconsistencies between observations made with mAb SC35, and transgenic SRSF2 constructs in the same experiment are noted. Intrigued by these disparities, we carried out a systematic re-characterization of this monoclonal antibody and its cellular targets. Unexpectedly, we found no evidence for SC35 mAb recognizing SRSF2 in human cells. In contrast, our results show that the 35 kDa namesake protein recognized by SC35 mAb is SRSF7, a related but distinct SR protein. More importantly, using mass-spectrometry, CRISPR-mediated knock-ins, immunoblotting and fluorescence microscopy, we show that the main target of SC35 mAb is SRRM2, a large (300 kDa), spliceosome-associated protein with prominent intrinsically disordered regions (IDRs) that sharply localizes to NS. Analysis of protein length evolution among metazoa revealed a peculiar IDR extension specifically for SRRM2 and SON in vertebrates, a hallmark of condensate formation. Combining these results, we tested a long-standing question in the study of NS: whether NS are formed around a specific core, or various SR-proteins self-assemble into a phase-separated compartment without the need for a defined core. Here we show that, the elusive core of NS is formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS, while combined depletion of SON together with SRRM2, but not other NS associated factors, or depletion of SON in a cell line where IDRs of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.
2020-10-26 | PXD021814 | Pride
Project description:Nuclear speckles (NSs) are nuclear biomolecular condensates that are postulated to arise through liquid-liquid phase separation (LLPS), although the detailed underlying forces driving NS formation remain elusive. SRRM2 and SON are 2 non-redundant scaffold proteins for NSs. How each individual protein governs assembly of NS protein network and the functional relationship between SRRM2 and SON are largely unknown. Here, we uncover immiscible multiphase of SRRM2 and SON within NSs. SRRM2 and SON are functionally independent, specifically regulating alternative splicing of subsets of mRNA targets, respectively. We further uncover that SRRM2 forms multicomponent liquid phase in cells to drive NS subcompartmentalization, which is reliant on homotypic interaction and heterotypic non-selective protein-RNA complex coacervation-driven multicomponent LLPS. SRRM2 RS domains form high-order oligomers, and can be replaced by oligomerizable synthetic modules, the serine residues within the RS domains, however, play an irreplaceable role in fine-tuning the liquidity of NSs.
2024-01-31 | GSE242898 | GEO
Project description:SON and SRRM2 were depleted in U2OS and HeLa cells and infected with HIV-1. Similarly HEK293T cells were infected with HIV-1 virus.
2025-04-01 | GSE264152 | GEO
Project description:SON and SRRM2 were depleted in U2OS and HeLa cells and infected with HIV-1. Similarly HEK293T cells were infected with HIV-1 virus.
2025-04-01 | GSE264150 | GEO
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 | GSE235083 | GEO
Project description:HeLa cells were cultured in DMEM, supplemented with 10% (v/v) FCS and penicillin/streptomycin under 5% CO2 at 37C. For iCLIP, HeLa cells expressing GFP fusion proteins were induced with doxycycline to adjust the level of recombinant protein to the level of the endogenous counterpart and irradiated with 150 mJ/cm2 UV light (254 nm). The iCLIP cDNA libraries were sequenced with 50 bp on an Illumina HiSeq 2000 instrument. RNASeq was performed as a control with 50 bp paired-end on an Illumina HiSeq 2000 instrument.
2016-07-28 | E-MTAB-4215 | biostudies-arrayexpress
Project description:Srrm2 splicing factor is a novel gene implicated in developmental disorders and diseases. However, the role of Srrm2 in early mammalian development remains unexplored. Here, we show that Srrm2 expression dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness characterized by the coexistence of cells expressing naive and formative markers, together with large gene expression shifts, including in serum-response transcription factor targets and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells identified splicing misregulation of specific genes, often linked to exon skipping. These results show that Srrm2 dosage is key in controlling stemness and cell fate decisions. Our findings unveil Srrm2’s molecular and cellular implications in development, shedding light on the involvement of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.
2024-04-12 | GSE243431 | GEO
Project description:Srrm2 splicing factor is a novel gene implicated in developmental disorders and diseases. However, the role of Srrm2 in early mammalian development remains unexplored. Here, we show that Srrm2 expression dosage is critical for maintaining embryonic stem cell pluripotency and cell identity. Srrm2 heterozygosity promotes loss of stemness characterized by the coexistence of cells expressing naive and formative markers, together with large gene expression shifts, including in serum-response transcription factor targets and differentiation-related genes. Depletion of Srrm2 by RNA interference in embryonic stem cells identified splicing misregulation of specific genes, often linked to exon skipping. These results show that Srrm2 dosage is key in controlling stemness and cell fate decisions. Our findings unveil Srrm2’s molecular and cellular implications in development, shedding light on the involvement of splicing regulators in early embryogenesis, developmental diseases and tumorigenesis.
2024-04-12 | GSE243429 | GEO
Project description:Nuclear speckles (NS) are among the most prominent biomolecular condensates. Despite their prevalence, research on the function of NS is virtually restricted to colocalization analyses, since an organizing core, without which NS cannot form, remains unidentified. The monoclonal antibody SC35, raised against a spliceosomal extract, is frequently used to mark NS. Unexpectedly, we found that this antibody was mischaracterized and the main target of SC35 mAb is SRRM2, a spliceosome-associated protein that sharply localizes to NS. Here we show that, the core of NS is likely formed by SON and SRRM2, since depletion of SON leads only to a partial disassembly of NS, while co-depletion of SON and SRRM2 or depletion of SON in a cell-line where intrinsically disordered regions (IDRs) of SRRM2 are genetically deleted, leads to a near-complete dissolution of NS. This work, therefore, paves the way to study the role of NS under diverse physiological and stress conditions.
| S-EPMC7671692 | biostudies-literature