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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE153803 | GEO | 2020/10/22

REPOSITORIES: GEO

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Project description:T cell activation leads to dramatic changes in cellular phenotype. We used CD3/CD28-activated human CD4 T cells to study how RNA binding proteins define the post-transcriptional landscape. Using RIPseq, we identified the RNA interactome of U2AF2 and show at the global level that U2AF2 binds the majority of transcripts that are differentially expressed and/or alternatively spliced during CD4 T cell activation. A unique protein interactome centered on U2AF2 is assembled in response to activation. Knocking down specific U2AF2 interacting partners (U2AF1, SYNCRIP, SRRM2, ILF2) selectively affects cytokine secretion and expression of activation markers. Furthermore, the expression and/or alternative splicing of transcripts important for immune cell function are also affected by knocking down these U2AF2 interacting proteins. U2AF1 and SYNCRIP knockdowns affect the proteins and transcripts bound to U2AF2, altering the transcriptome. Our work highlights the importance of RNA binding protein complexes in regulating the differential expression and alternative splicing that defines T cell activation. HTA 2.0 microarray results of total from a primary CD4 T cell culture after treatment with siRNAs (Control, U2AF1, SRRM2, SYNCRIP, and ILF2) and 24 hours after anti-CD3/CD28 bead activation Please note that the Series supplementary 'TotalRNA_HTA2_all_siRNA_100414.xslx' file has multiple worksheets containing: 1) Differentially expressed genes in ctrl siRNA vs. U2AF1 siRNA; 2) Differntially spliced genes in ctrl siRNA vs. U2AF1 siRNA; 3) Differentially spliced exons in ctrl siRNA vs. U2AF1 siRNA; 4) Differentially expressed genes in ctrl siRNA vs. SRRM2 siRNA; 5) Differntially spliced genes in ctrl siRNA vs. SRRM2 siRNA; 6) Differentially spliced exons in ctrl siRNA vs. SRRM2 siRNA; 7) Differentially expressed genes in ctrl siRNA vs. SYNCRIP siRNA; 8) Differntially spliced genes in ctrl siRNA vs. SYNCRIP siRNA; 9) Differentially spliced exons in ctrl siRNA vs. SYNCRIP siRNA; 10) Differentially expressed genes in ctrl siRNA vs. ILF2 siRNA; 11) Differntially spliced genes in ctrl siRNA vs. ILF2 siRNA; 12) Differentially spliced exons in ctrl siRNA vs. ILF2 siRNA

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

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