PDGFRa signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking
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ABSTRACT:
PROVIDER: PRJNA1096027 | ENA |
REPOSITORIES: ENA
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Project description:Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRa signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRa signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRa signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRa signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRa signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.
2024-05-02 | GSE263168 | GEO
Project description:Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) plays a critical role in craniofacial development, as mutations in PDGFRA are associated with cleft lip/palate in humans and Pdgfra mutant mouse models display varying degrees of facial clefting. Phosphatidylinositol 3-kinase (PI3K)/Akt is the primary effector of PDGFRa signaling during skeletal development in the mouse. We previously demonstrated that Akt phosphorylates the RNA-binding protein serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRa signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to its nuclear translocation. We further showed that ablation of Srsf3 in the murine neural crest lineage results in severe midline facial clefting, due to defects in proliferation and survival of cranial neural crest cells, and widespread alternative RNA splicing (AS) changes. Here, we sought to determine the molecular mechanisms by which Srsf3 activity is regulated downstream of PDGFRa signaling to control AS of transcripts necessary for craniofacial development. We demonstrated via enhanced UV-crosslinking and immunoprecipitation (eCLIP) of MEPM cells that PDGF-AA stimulation leads to preferential binding of Srsf3 to exons and loss of binding to canonical Srsf3 CA-rich motifs. Through the analysis of complementary RNA-seq data, we showed that Srsf3 activity results in the preferential inclusion of exons with increased GC content and lower intron to exon length ratio. Moreover, we found that the subset of transcripts that are bound by Srsf3 and undergo AS upon PDGFRa signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRa signaling leads to retention of the receptor in early endosomes and increases in downstream PI3K-mediated Akt signaling. Taken together, our findings reveal that growth factor-mediated phosphorylation of an RNA-binding protein underlies gene expression regulation necessary for mammalian craniofacial development.
2024-05-02 | GSE263169 | GEO
Project description:PDGFRa signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking (eCLIP)
| PRJNA1096029 | ENA
Project description:PDGFRa signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking (RNA-seq)
| PRJNA1096030 | ENA
Project description:Signaling through the platelet-derived growth factor receptor alpha (PDGFRa) is critical for mammalian craniofacial development, though the mechanisms by which the activity of downstream intracellular effectors is regulated to mediate gene expression changes have not been defined. We find that the RNA-binding protein Srsf3 is phosphorylated at Akt consensus sites downstream of PI3K-mediated PDGFRa signaling in palatal mesenchyme cells, leading to its nuclear translocation. We further demonstrate that ablation of Srsf3 in the neural crest lineage leads to facial clefting due to defective cranial neural crest cell specification and survival. Finally, we show that Srsf3 regulates the alternative RNA splicing of transcripts encoding protein kinases in the facial process mesenchyme to negatively regulate PDGFRa signaling. Collectively, our findings reveal that PI3K/Akt-mediated PDGFRa signaling primarily modulates gene expression through alternative RNA splicing in the facial mesenchyme and identify Srsf3 as a critical regulator of craniofacial development.
2021-06-30 | GSE161510 | GEO
Project description:This SuperSeries is composed of the SubSeries listed below.
2024-05-02 | GSE263170 | GEO
Project description:Loss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer’s disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD’s cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell derived neurons (hiPSC-Ns) to test this hypothesis. In SORL1 deficient (SORL1KO) cell lines, we map the trafficking of the glutamate receptor and the BDNF neurotrophic receptor, two kinds of transmembrane proteins that depend on endosomal recycling and that are linked to AD pathophysiology. We find that as with APP, SORL1 is required for efficient endosomal recycling of the glutamate receptor AMPA1 (GLUA1) and the BDNF receptor Tropomyosin-related kinase B (TRKB). Next, we used cell lines engineered to overexpress SORL1 and find that increased SORL1 expression enhances recycling for APP and GLUA1. Finally, we performed an unbiased transcriptomic screen of SORL1KO neurons and the data further support SORL1’s role in endosomal recycling. We observed altered expression networks that regulate cell surface trafficking and neurotrophic signaling. Collectively, and together with other recent observations, these findings suggest that SORL1 is a key and broad regulator of retromer-dependent endosomal recycling in neurons, a conclusion that has both pathogenic and therapeutic implications.
2022-03-01 | GSE180793 | GEO
Project description:Trafficking of G protein-coupled receptors (GPCRs) through the endo-lysosomal pathway is critical to homeostatic regulation of GPCRs following activation with agonist. Identifying the genes involved in GPCR trafficking is challenging due to the complexity of sorting operations and low affinity protein-receptor interactions. Here we show that the engineered enzyme APEX2 is a highly sensitive biosensor for GPCR trafficking to the lysosome, and this trafficking can be monitored through APEX-based activation of fluorogenic substrates such as Amplex UltraRed (AUR). We used GPCR-APEX2/AUR to perform a genome-wide CRISPR interference screen focused on the delta type opioid receptor (DOR), a GPCR which modulates anxiety and pain. We provide a genetic map of components in the endo-lysosomal pathway necessary for DOR trafficking to the lysosome and subsequent downregulation. Using our GPCR-APEX2 pipeline, we demonstrate that a gene identified in our screen, DNAJC13, controls trafficking of DOR to the lysosome by regulating the endosomal proteome and endosomal homeostasis.
2024-07-01 | GSE242366 | GEO
Project description:Srsf3 mediates alternative RNA splicing downstream of PDGFRα signaling.
| PRJNA678558 | ENA
Project description:Membrane trafficking is defined as the vesicular transport of molecules throughout the cell. In intestinal enterocytes, defects in endocytic/recycling pathways impair their function and are linked to genetic diseases. How does trafficking regulate intestinal tissue homeostasis is poorly understood. Using the Drosophila intestine as an in vivo model system, we investigated enterocyte-specific functions for early endosomal trafficking in gut homeostasis. We focused on the small GTPase Rab21 that regulates specific early endosomal trafficking steps. Rab21-depleted guts showed severe intestinal morphology abnormalities, with deregulated homeostasis associated with a gain in mitotic cells and increased cell death. Increased in both apoptosis and Yki signaling were responsible for compensatory proliferation and tissue inflammation. Using a RNAi screen, we identified autophagy and specific membrane trafficking regulators phenocopying Rab21 loss. We further showed that Rab21-induced hyperplasia was rescued by inhibition of Egfr signaling, and we identified improperly trafficked cargoes in enterocytes depleted of Rab21. Our data shed light on an important role for the early endosomal protein Rab21, and early endosomal trafficking in enterocytes-mediated intestinal homeostasis.
2022-08-12 | PXD022413 | Pride