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Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis

ABSTRACT: Purpose: The goals of this study are to compare transcriptomes using RNA-seq of mouse myoblasts (C2C12 cell line) in undifferentiated and differentiated states and with siRNA-mediated knock down of the RNA binding proteins, Rbfox1 (only expressed in differentiated state) and Rbfox2 (expressed in both undifferentiated and differentiated states). Methods: Differentiated and undifferentiated C2C12 cultures treated with Rbfox1 (differentiated only) or Rbfox2 siRNAs or a mock siRNA transfection were used for RNA-Seq analysis using Illumina HiSeq2000. 101x2 paired-end RNA-seq reads were first uniquely aligned to the mouse genome (mm9) using TopHat 1.4.1. RSEM was used to count the number of reads mapped to genes using UCSC database, followed by edgeR to call differentially expressed genes with false discovery rate less than 0.01. Cufflinks was used to reconstruct isoforms and analyze alternative splicing and percent spliced in (PSI) was calculated. PSI values were validated by RT-PCR. Results: 58-88% of the RNA-seq reads from technical and biological replicates mapped uniquely to the mouse genome. Analysis of gene expression and alternative splicing changes are published in Singh et al. Molecular Cell (2014). Conclusions: Our study has identified gene expression and alternative splicing transitions that occur during myoblast differentiation, demonstrate that 30% of the splicing transitions are regulated by Rbfox2, demonstrated that Rbfox2 is required for a late step of myoblast differentiation and identified two Rbfox2-regulated splicing transitions that are required for differentiation.

ORGANISM(S): Mus musculus

PROVIDER: GSE58928 | GEO | 2014/07/15

SECONDARY ACCESSION(S): PRJNA253937

REPOSITORIES: GEO

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Similar Datasets

Rbfox2-coordinated alternative splicing of Mef2d and Rock2 controls myoblast fusion during myogenesis

Project description:Purpose: The goals of this study are to compare transcriptomes using RNA-seq of mouse myoblasts (C2C12 cell line) in undifferentiated and differentiated states and with siRNA-mediated knock down of the RNA binding proteins, Rbfox1 (only expressed in differentiated state) and Rbfox2 (expressed in both undifferentiated and differentiated states). Methods: Differentiated and undifferentiated C2C12 cultures treated with Rbfox1 (differentiated only) or Rbfox2 siRNAs or a mock siRNA transfection were used for RNA-Seq analysis using Illumina HiSeq2000. 101x2 paired-end RNA-seq reads were first uniquely aligned to the mouse genome (mm9) using TopHat 1.4.1. RSEM was used to count the number of reads mapped to genes using UCSC database, followed by edgeR to call differentially expressed genes with false discovery rate less than 0.01. Cufflinks was used to reconstruct isoforms and analyze alternative splicing and percent spliced in (PSI) was calculated. PSI values were validated by RT-PCR. Results: 58-88% of the RNA-seq reads from technical and biological replicates mapped uniquely to the mouse genome. Analysis of gene expression and alternative splicing changes are published in Singh et al. Molecular Cell (2014). Conclusions: Our study has identified gene expression and alternative splicing transitions that occur during myoblast differentiation, demonstrate that 30% of the splicing transitions are regulated by Rbfox2, demonstrated that Rbfox2 is required for a late step of myoblast differentiation and identified two Rbfox2-regulated splicing transitions that are required for differentiation. Undifferentiated and differentiated C2C12 cultures with Rbfox2 depletion or Rbfox1 depletion (differentiated only) in at least duplicate samples analyzed by deep sequencing on Illumina HiSeq2000.

2014-07-15 | E-GEOD-58928 | biostudies-arrayexpress

C2C12 myoblast vs myotube

Project description:Comparison of undifferentiated C2C12 myoblast to 4 day differentiated myotubes. Keywords: other

2005-09-07 | GSE3243 | GEO

Transcription profiling of mouse undifferentiated C2C12 myoblast to 4 day differentiated myotubes

Project description:Comparison of undifferentiated C2C12 myoblast to 4 day differentiated myotubes. Experiment Overall Design: this experiment include 2 samples and 6 replicates

2008-06-12 | E-GEOD-3243 | biostudies-arrayexpress

DMD-targeted RNA-seq data in C25Cl48 muscular cell line treated with siRNA targeting 16 different RBPs

Project description:Purpose: The goal of this study was to identify the RBPs that regulate the inclusion of the exons in the muscular isoform of the DMD gene (Dp427m). We compared the splicing pattern of the Dp427m from C25Cl48 treated with a control siRNA or siRNA targeting 16 different RBPs. Method: The C25Cl48 were treated by RNAi in duplicates (or n=4 for the control condition) and submitted to 3 days of myogenic differentiation. The RBPs tested were CELF1, DAZAP1, DDX5/DDX17, hnRNPA1, hnRNPA2, MBNL1, PTBP1, QKI, RBFOX1, RBFOX2, RBM4, SRSF1, SRSF2, TDP-43 and Tra2b. The silencing efficiency was controlled by RT-qPCR and Western blotting analysis. RNA was reverse transcribed using oligodT and the Dp427m (11.3kb) coding sequence was amplified by Long-Range PCR and sequenced on a 454 GS junior platform. Reads that passed quality filters were mapped to the human X chromosome with STAR. The Percent-Spliced-In (PSI) was calculated for each splicing event using intron-centric metrics (Pervouchine et al., 2013), with the SJPIPE pipeline, from the ipsa package. Junctions were filtered to keep only those covered by a minimum of 5 reads in at least one out of the two biological replicates or 2/4 in the control condition. Results: Using this DMD-targeted RNA-seq approach, we obtained an average read depth of 1638X, allowing reliable detection of alternative splicing events. Based on a cutoff of |[delta]PSI|≥0.05, we identified 8 exons that were deregulated in at least one of the RBPs tested, and 15 RNAi conditions induced splicing changes. Conclusion: The 8 deregulated exons identified here are mainly exons that were previously found alternatively spliced in other dystrophin isoforms.

2020-10-22 | GSE153803 | GEO

RBFOX1 Splicing and Transcriptional Regulation in Neurons

Project description:We used RNA sequencing to identify the RBFOX1 splicing network at a genome-wide level in primary human neural stem cells during differentiation. We observe that RBFOX1 regulates a large set of alternative splicing events implicated in neurogenesis and cell maintenance. Subsequent alterations in gene expression define an additional transcriptional network regulated by RBFOX1 involved in neurodevelopmental pathways remarkably parallel to those affected by splicing. RNA sequencing at a 75bp single-end read scale was performed using polyA-enriched RNA from 5 biological replicates of primary human neural progenitor cell lines generated by lentiviral-mediated knockdown of GFP (control) or RBFOX1 and differentiated for 4 weeks.

2012-06-28 | E-GEOD-36710 | biostudies-arrayexpress

Alternative splicing outcomes across an RNA-binding protein concentration gradient

Project description:Alternative splicing (AS) is a dynamic RNA processing step that produces multiple RNA isoforms from a single pre-mRNA transcript and contributes to the complexity of the cellular transcriptome and proteome. This process is regulated through a network of cis-regulatory sequence elements and trans-acting factors, most-notably RNA binding proteins (RBPs). The muscleblind-like (MBNL) and RNA binding fox-1 homolog (RBFOX) are two well characterized families of RBPs that regulate fetal to adult AS transitions critical for proper muscle, heart, and central nervous system development. To better understand how the concentration of these RBPs influences AS transcriptome wide, we engineered an MBNL1 and RBFOX1 inducible HEK-293 cell line. We found that induction of exogenous RBFOX1 modulated AS outcomes, despite significant levels of endogenous RBFOX1 and RBFOX2 expression. We characterized AS outcomes by RNAseq following dose-dependent MBNL1 induction to generate transcriptome wide dose-response curves of skipped exon events. Analysis of 43 validated splicing events by RT-PCR revealed a spectrum of dose-response splicing curves with a 2-fold range for EC50 values and slope values ranging from 4 to 10. Analysis of this data suggests that exclusion events, when compared to inclusion events, regulated by MBNL1, may require higher protein concentrations to regulate, and that multiple arrangements of YGCY motifs produce similar splicing outcomes. Together these data suggest that rather than a simple association between the configuration of RBP binding sites and a specific splicing outcome, that a complex network governs both AS inclusion and exclusion events that operates in an RBP dose-dependent manner.

2023-01-01 | GSE221838 | GEO

Rbfox2 controls autoregulation in RNA binding protein networks

Project description:We generated a global analysis of Rbfox2 splicing regulation combined with a highly specific, single nucleotide-resolution Rbfox2 RNA binding map. We found that Rbfox2 regulates the splicing and expression of many previously unknown targets, and particularly a number of RNA binding proteins (RBPs), by modulating alternative splicing coupled-NMD. Based on our observations of RBP-Rbfox2 co-regulation with a polarity predicted by Rbfox2 binding, we propose a model whereby Rbfox2 tunes autoregulatory splicing events to control RBP expression levels and in turn alter their respective splicing networks. iCLIP for epitope-tagged Rbfox2 and control untagged Rbfox2; RNAseq of control and Rbfox2 knockdown in mouse embryonic stem cells

2014-03-17 | E-GEOD-54794 | biostudies-arrayexpress

Machine learning inference of continuous single-cell state transitions during myoblast differentiation and fusion

Project description:Cells dynamically change their internal organization via continuous cell state transitions to mediate a plethora of physiological processes. Understanding such continuous processes is severely limited due to a lack of tools to measure the holistic physiological state of single cells undergoing a transition. We combined live-cell imaging and machine learning to quantitatively monitor skeletal muscle precursor cell (myoblast) differentiation during multinucleated muscle fiber formation. Our machine learning model predicted the continuous differentiation state of single primary murine myoblasts over time and revealed that inhibiting ERK1/2 leads to a gradual transition from an undifferentiated to a terminally differentiated state 7.5-14.5 hours post inhibition. Myoblast fusion occurred ~3 hours after predicted terminal differentiation. Moreover, we showed that our model could predict that cells have reached terminal differentiation under conditions where fusion was stalled, demonstrating potential applications in screening. This method can be adapted to other biological processes to reveal connections between the dynamic single-cell state and virtually any other functional readout.

2024-05-23 | PXD047198 | Pride

Transcription profiling of human 293FT-human embryonic kidney cells, undifferentiated FM95-14 myoblast, and differentiated myoblast to verify FM95-14 expression

Project description:Undifferentiated FM95-14 human embryonic myoblasts contain myogenic stem/progenitor cells. Gene expression analysis and comparison to FM95-14 cells that have been allowed to differentiate will allow the ascertainment of genes that are implicated in myogenic lineage commitment. Human myoblast were also differentiated for 4 days. 293FT-human embroynal kidney were used as a human control cell line for human myoblast study. Experiment Overall Design: this experiment include 3 samples (293FT-human embroynal kidney, undifferentiated FM95-14 myoblast, and differentiated myoblast) with 3 replicates on 2 platforms for a total of 18 Samples.

2008-04-09 | E-GEOD-10435 | biostudies-arrayexpress

Expression and alternative splicing data from 1-month-old Rbfox1 KO brain

Project description:Rbfox1 regulates the alternative splicing of many transcripts in neurons. We have characterized the Rbfox1-dependent changes in expression and alternative splicing by comparing Rbfox1-KO brain to WT brain. In this dataset, we include the splicing and expression data obtained from dissected WT and Rbfox1 KO mouse brains. 6 total samples were analyzed: brains from 3 WT male mice and 3 Rbfox1 KO male mice, all 1 month of age.

2011-05-16 | E-GEOD-28421 | biostudies-arrayexpress

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