Deciphering RBP - alternative splicing networks in ALS iPSC-MN: NOVA1 gain and loss of function systems
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PROVIDER: EGAS00001005881 | EGA |
REPOSITORIES: EGA
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Project description:3 control iPSC lines differentiated into iPSC-derived motor neurons transduced with either EGFP or NOVA1 lentivirus.
| EGAD00001008423 | EGA
Project description:We produced enhanced cross-linking immunoprecipitation (eCLIP) sequencing data of the RNA binding proteins (RBP) TDP-43, NOVA1, NOVA2 and RBFOX2 in iPSC motor neurons of two healthy control individuals, respectively.
| EGAS00001005880 | EGA
Project description:Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the M-bM-^@M-^\neuron specificM-bM-^@M-^] Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signalling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1. 3 batch of primary rat pancreatic beta cells were examined under 2 conditions: control and with Nova1 splicing factor knock-down
2014-10-01 | E-GEOD-59633 | biostudies-arrayexpress
Project description:Alternative splicing (AS) is a fundamental mechanism for the regulation of gene expression. It affects more than 90% of human genes but its role in the regulation of pancreatic beta cells, the producers of insulin, remains unknown. Our recently published data indicated that the “neuron specific” Nova1 splicing factor is expressed in pancreatic beta cells. We have presently coupled specific knockdown (KD) of Nova1 with RNA-sequencing to determine all splice variants and downstream pathways regulated by this protein in beta cells. Nova1 KD altered the splicing of nearly 5000 transcripts. Pathway analysis indicated that these genes are involved in exocytosis, apoptosis, insulin receptor signalling, splicing and transcription. In line with these findings, Nova1 silencing inhibited insulin secretion and induced apoptosis basally and after cytokine treatment in rodent and human beta cells. These observations identify a novel layer of regulation of beta cell function, namely AS controlled by key splicing regulators such as Nova1.
2014-10-01 | GSE59633 | GEO
Project description:iPSC-derived motor neurons from 5 NOVA1 knock out and 5 NOVA1 wt lines in the CVB background.
| EGAD00001008427 | EGA
Project description:To discover RBPs with increased insolubility in a human ALS model, we applied a well established dual-SMAD inhibition-based protocol (Fang et al., 2019; Markmiller et al., 2021; Markmiller et al., 2018; Martinez et al., 2016) to generate iPSC-MN from six control iPSC lines, from four iPSC lines originating from two sALS patients, and from two iPSC lines originating from fALS patients with pathogenic variants in the TARDBP gene (Table S1; Figure S1A). No difference in differentiation capacity was observed (Figure S1B-G), resulting in average 40% ISL1+ MN (Figures S1G), comparable to numbers observed in large scale MN differentation studies (Baxi et al., 2022). The susceptibility of ALS MN to sodium arsenite-induced stress was not changed (Figure S1H and I). Next, we asked which proteins exhibit an increased insolubility in our ALS iPSC-MN. We fractioned iPSC- MN by lysis in radio-immunoprecipitation assay (RIPA) buffer, followed by ultracentrifugation and solubilization of RIPA insoluble proteins in urea buffer. The ultracentrifugation-cleared RIPA insoluble protein fraction is widely used to study protein insolubility in the context of neurodegeneration (Nuber et al., 2013; Walker et al., 2015). Label-free mass spectrometry of the insoluble protein fraction was utilized to identify proteins that are insoluble in sALS and fALS, relative to control iPSC-MNs (Figure 1A). Gene ontology (GO) analysis of the 100 proteins (top 2.9% of all detected proteins) with the highest label free quantification (LFQ) intensities in controls (Figure S1J) revealed that ‘unfolded protein binding’ (corrected P = 7.95 x 10-16) and ‘structural constituent of cytoskeleton’ (corrected P = 1.47 x 10-10) were among the 10 most significantly enriched GO terms, indicating enrichment of insoluble proteins (Figure S1K). Principle component analysis of the insoluble fractions did not distinguish ALS from control samples, suggesting that the overall insoluble proteome is not changed (Figure S1L). At threshold P ≤ 0.05 (Welch’s t-test) and fold change ≥ 1.5, we identified 88 proteins enriched in the insoluble fraction in ALS samples relative to control (Figure 1B). When the sample labels were randomly shuffled, we observed an average of 7.5 proteins (~12-fold lower) as differentially enriched at the same statistical thresholds, indicative of an ALS-specific protein insolubility pattern (Figure 1C). The 88 candidate proteins included cytoskeletal components and motor proteins, functional categories associated with prominent ALS in vitro phenotypes (Akiyama et al., 2019; Egawa et al., 2012; Fazal et al., 2021; Guo et al., 2017; Kreiter et al., 2018) (Figure 1D). Notably, 5 RBPs, NOVA1, ELAVL4, FXR2, RBFOX2, and RBFOX3 were also enriched (Figure 1D). The NOVA1 paralog NOVA2 was significantly enriched (P = 0.03) but did not meet our enrichment threshold (fold change = 1.36). Interestingly, insoluble TDP-43 protein was not significantly different in ALS and control (P = 0.98; fold change = 0.97). Western blot analysis confirmed the increase in insolubility of NOVA1, NOVA2, ELAVL4, RBFOX2 and RBFOX3 (Figure 1E and 1F). The soluble protein levels of NOVA1 and NOVA2 were also increased (Figure 1F). In conclusion, we identified 5 RBPs with elevated insoluble protein levels of ALS-iPSC-MNs.
2022-06-28 | PXD032140 | Pride
Project description:To elucidate the function of Nova1 in neurons, especially in inhibitory neurons, gene expression profiling analysis (RNAseq, TRAP-seq) was performed using Nova1 conditional knockout mice and their primary neurons.
2024-08-15 | GSE212473 | GEO
Project description:We produce RNA-seq datasets of iPSC-derived motor neurons (iPSC-MN) from healthy controls and sporadic ALS patients and controls and familial ALS patients with pathogenic variants in TARDBP.
| EGAS00001005879 | EGA
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ progenitors. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We used Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 to help identify GRHL2 target genes and their functions in HBE cells. Primary HBE cells from three donors were infected with modified TripZ lentivirus designed to express EGFP or DN-GRHL2 in response to Dox. Dox was added to cells cultured at the air-liquid interface from day 7 to 14.
2013-05-08 | E-GEOD-44807 | biostudies-arrayexpress
Project description:The airways of the human lung are lined by an epithelium made up of ciliated and secretory luminal cells and undifferentiated p63+ Krt5+ basal cells. The integrity of this epithelium and its ability to act as a selective barrier are critical for normal lung function. In other epithelia there is evidence that transcription factors of the evolutionarily conserved grainyheadlike (GRHL) family play key roles in co-ordinating the expression of numerous proteins required for epithelial morphogenesis, differentiation, remodeling and repair. However, little is known about their function in the adult lung. We use Affymetrix microarray analysis to compare transcripts in lentivirus transfected primary human bronchial epithelial (HBE) cells expressing either EGFP or DN-GRHL2 for 48h when the transepithelial electrical resistance (TER) reached a threshold level. The goal is to identify direct target genes of GRHL2 and early events in the uncoupling of junctional interactions, including those regulating transepithelial resistance. Primary HBE cells from three donors were infected with EGFP or DN-GRHL2 expression lentivirus. Dox was added for 48h to induce the expression of either EGFP or DN-GRHL2 when TER reached a threshold level in ALI culture.
2013-05-08 | E-GEOD-44808 | biostudies-arrayexpress