Modulation of insulin secretion by RBFOX2-mediated alternative splicing

Moss, Nicole D.; Wells, Kristen L.; Theis, Alexandra; Kim, Yong-Kyung; Spigelman, Aliya F.; Liu, Xiong; MacDonald, Patrick E.; Sussel, Lori

doi:10.1038/s41467-023-43605-4

Cited by 10 publications

(2 citation statements)

References 73 publications

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“…Conversely, Srsf3-mediated AS may be delayed following transcript binding in the short timeframe of our experimental design. However, the extent of overlap that we observed is in line with previous studies correlating alternatively-spliced transcripts upon knockdown of an RBP with endogenous eCLIP results for that same RBP, including Rbfox2 (10%) (Moss et al, 2023) and LUC7L2 (18-26%) (Jourdain et al, 2021). The degree to which our RNA-seq and eCLIP datasets overlapped here points to the robustness and biological significance of our findings.…”

Section: Discussionsupporting

confidence: 92%

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

Forman,

Sajek,

Larson

et al. 2024

Preprint

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Signaling through the platelet-derived growth factor receptor alpha (PDGFRα) 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 PDGFRα 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 PDGFRα 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 PDGFRα 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 PDGFRα signaling commonly encode regulators of PI3K signaling and early endosomal trafficking. Functional validation studies further confirmed that Srsf3 activity downstream of PDGFRα 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.

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Section: Discussionsupporting

confidence: 92%

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

Forman,

Sajek,

Larson

et al. 2024

Preprint

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“…Recent findings further implicate RBFOX2 in insulin regulation through splicing targets involved in exocytosis, and pancreas-specific Rbfox2 mutant male mice exhibit impaired glucose tolerance and insulin secretion (Moss et al, 2023). Intriguingly, Rbfox1 and Rbfox2 silencing in rat insulinoma beta cell line led to upregulation of insulin secretion following glucose stimulation, which was dose-dependent and based on actin remodelling changes in Rbfox1 knockdown (Juan-Mateu et al, 2017).…”

Section: Rbfox2mentioning

confidence: 96%

Translational study on the role of genetic and prenatal risk factors in neurodevelopmental psychiatric disorders

Yotova

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Neurodevelopmental psychiatric disorders (NPDs) like attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), and schizophrenia, affect millions of people worldwide. Despite recent progress in NPD research, much remains to be discovered about their underpinnings, therapeutic targets, effects of biological sex and age. Risk factors influencing brain development and signalling include prenatal inflammation and genetic variation. This dissertation aimed to build upon these findings by combining behavioural, molecular, and neuromorphological investigations in mouse models of such risk factors, i.e. maternal immune activation (MIA), neuron-specific overexpression (OE) of the cytoplasmatic isoforms of the RNA-binding protein RBFOX1, and neuronal deletion of the small Ras GTPase DIRAS2. Maternal infections during pregnancy pose an increased risk for NPDs in the offspring. While viral-like MIA has been previously established elsewhere, this study was the first in our institution to implement the model. I validated NPD-relevant deficits in anxiety- and depression-like behaviours, as well as dose- and sex-specific social deficits in mouse offspring following MIA in early gestation. Proteomic analyses in embryonic and adult hippocampal (HPC) synaptoneurosomes highlighted novel and known targets affected by MIA. Analysis of the embryonic dataset implicated neurodevelopmental disruptions of the lipid, polysaccharide, and glycoprotein metabolism, important for proper membrane function, signalling, and myelination, for NPD-pertinent sequelae. In adulthood, the observed changes encompassed transmembrane trafficking and intracellular signalling, apoptosis, and cytoskeletal organisation pathways. Importantly, 50 proteins altered by MIA in embryonic and adult HPC were enriched in the NPD-relevant synaptic vesicle cycle. A persistently upregulated protein cluster formed a functional network involved in presynaptic signalling and proteins downregulated in embryos but upregulated in adults by MIA were correlated with observed social deficits. 49/50 genes encoding these proteins were significantly associated with NPD- and comorbidity-relevant traits in human phenome-wise association study data for psychiatric phenotypes. These findings highlight NPD-relevant targets for future study and early intervention in at-risk individuals. MIA-evoked changes in the neuroarchitecture of the NPD-relevant HPC and prefrontal cortex (PFC) of male and female mice highlighted sex- and region-specific alterations in dendritic and spine morphology, possibly underlining behavioural phenotypes. To further investigate genetic risk factors of NPDs, I performed a study based on the implications of RBFOX1’s pleiotropic role in neuropsychiatric disorders and previous preclinical findings. Cytoplasmatic OE of RBFOX1, which affects the stability and translation of thousands of targets, was used to disseminate its role in morphology and behaviour. RBFOX1 OE affected dendritic length and branching in the male PFC and led to spine alterations in both PFC and HPC. Due to previously observed ASD-like endophenotypes in our Rbfox1 KO mice and the importance of gene × environment effects on NPD susceptibility, I probed the interaction of cytoplasmatic OE and a low-dose MIA on offspring. Both RBFOX1 OE alone and with MIA led to increased offspring loss during the perinatal period. Preliminary data suggested that RBFOX1 OE × MIA might increase anxiety- and anhedonia-like behaviours. Morphological changes in the adult male OE HPC and PFC suggested increased spine density and reduced dendritic complexity. A small post-mortem study in human dorsolateral PFC of older adults did not reveal significant effects of a common risk variant on RBFOX1 abundance. To expand upon NPD genetic risks, I evaluated the effects of a homo- (KO) or heterozygous (HET) Diras2 deletion in a novel, neuron-specific mouse model. DIRAS2’s function is largely unknown, but it has been associated with ADHD in humans and neurodevelopment in vitro. In adult mice, there were subtle sex-specific effects on behaviour, i.e. more pronounced NPD-relevant deficits in males, in keeping with human data. KO mice had subtly improved cognitive performance, while HET mice exhibited behaviours in line with core ADHD symptoms, e.g. earning difficulties (females), response inhibition deficits and hyperactivity (males), suggesting Diras2 dose-sensitivity and sex-specificity. The morphological findings revealed multiple aberrations in dendritic and spine morphology in the adult PFC, HPC, and amygdala of HET males. KOs changes in spine and dendritic morphology were exclusively in the PFC and largely opposite to those in HETs and NPD-like phenotypes. Region- and genotype-specific expression changes in Diras2 and Diras1 were observed in six relevant brain regions of adult HET and KO females, also revealing differences in the survival and morphology regulator mTOR, which might underlie observed differences. In conclusion, the effects of MIA and partial Diras2 knockdown resembled each other in core, NPD-associated behavioural and morphological phenotypes, while cytoplasmatic RBFOX1 OE and full Diras2 KO differed from those. My findings suggest complex dose- and sex-dependent relationships between these prenatal and genetic interventions, whose NPD-relevant influences might converge onto neurodevelopmental molecular pathways. An assessment of such putative overlap, based on available data from the MIA proteomic analyses of embryonic and adult HPC, suggested the three models might be linked via downstream targets, interactions, and upstream regulators. Future studies should disseminate both distinct and shared aspects of MIA, RBFOX1, and DIRAS2 relevant to NPDs and build upon these findings.

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PARN Maintains RNA Stability to Regulate Insulin Maturation and GSIS in Pancreatic β Cells

Xie,

Chen,

Wang

et al. 2024

Advanced Science

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Diabetes, a metabolic disorder characterized by hyperglycemia, underscores the importance of normal pancreatic β‐cell development and function in maintaining glucose homeostasis. Poly(A)‐specific ribonuclease (PARN) serves as the principal regulator of messenger RNA (mRNA) stability, yet its specific role in pancreatic β cells remains unclear. This study utilizes mice with targeted PARN deficiency in β cells to elucidate this role. Notably, Parn conditional knockout mice present unaltered β‐cell development and insulin sensitivity but reduced glucose‐stimulated insulin secretion (GSIS). The observed outcomes are corroborated in NIT‐1 cells. Furthermore, transcriptomic analyses reveal aberrant mRNA expression of genes crucial for insulin secretion in PARN‐deficient β cells. Insights from linear amplification of complementary DNA ends and sequencing and coimmunoprecipitation experiments reveal an interaction between PARN and polypyrimidine tract‐binding protein 1 (PTBP1), regulating the RNA stability of solute carrier family 30, member 8 (Slc30a8) and carbohydrate sulfotransferase 3 (Chst3). Interference with either PARN or PTBP1 disrupts this stability. These data indicate that PARN deficiency hampers GSIS and insulin maturation by destabilizing Slc30a8 and Chst3 RNAs. These findings provide compelling evidence indicating that PARN is a potential therapeutic target for enhancing insulin maturation and secretion.

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Modulation of insulin secretion by RBFOX2-mediated alternative splicing

Cited by 10 publications

References 73 publications

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

PDGFRα signaling regulates Srsf3 transcript binding to affect PI3K signaling and endosomal trafficking

Translational study on the role of genetic and prenatal risk factors in neurodevelopmental psychiatric disorders

PARN Maintains RNA Stability to Regulate Insulin Maturation and GSIS in Pancreatic β Cells

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