Exon array analysis in the mouse myoblast cell line, C2C12 cells treated with control-, Cugbp1- or Mbnl1-siRNA
ABSTRACT: CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. Using HITS-CLIP anlysis, we found CUGBP1 and MBNL1 preferentially bind to alternatively spliced introns and exons, as well as to the 3' UTRs. To analyze more directly the role of CUGBP1/MBNL1 binding in alternative splicing, we performed exon array analysis in C2C12 cells using expression arrays. We analyzed total RNA of C2C12 cells treated with control-, Cugbp1- or Mbnl1-siRNA. RNA was harvested 48 hrs after transfection.
Project description:CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. Using HITS-CLIP anlysis, we found CUGBP1 and MBNL1 preferentially bind to alternatively spliced introns and exons, as well as to the 3' UTRs. To analyze more directly the role of CUGBP1/MBNL1 binding to the 3’ UTR, we performed global analysis of mRNA stability in C2C12 cells using expression arrays, and found that CUGBP1 and MBNL1 regulate decay of endogenous mRNAs. We analyzed total RNA of C2C12 cells treated with control-, Cugbp1- or Mbnl1-siRNA. We analyzed 3 time points after addition of actionmycin D (0, 2.5, 5 hours).
Project description:CUGBP1 and MBNL1 are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. Using HITS-CLIP anlysis, we found CUGBP1 and MBNL1 preferentially bind to alternatively spliced introns and exons, as well as to the 3' UTRs. To analyze more directly the role of CUGBP1/MBNL1 binding to the 3’ UTR, we performed global analysis of mRNA stability in C2C12 cells using expression arrays, and found that CUGBP1 and MBNL1 regulate decay of endogenous mRNAs. Overall design: We analyzed total RNA of C2C12 cells treated with control-, Cugbp1- or Mbnl1-siRNA. We analyzed 3 time points after addition of actionmycin D (0, 2.5, 5 hours).
Project description:CUG-binding protein 1 (CUGBP1) and muscleblind-like 1 (MBNL1) are developmentally regulated RNA-binding proteins that are causally associated with myotonic dystrophy type 1. We extensively determined RNA-binding sites of CUGBP1 and MBNL1 to investigate their roles in RNA processing. We also analyzed polypyrimidine tract-binding protein (PTB) as a control. CUGBP1 and MBNL1 preferentially bind to alternatively spliced introns and exons, respectively, and regulate alternative splicing events. Moreover, CUGBP1 and MBNL1 are preferentially bound to the 3' untranslated regions (UTRs), in particular of genes for RNA-binding proteins, and facilitate decay of the bound mRNAs. In addition, CUGBP1 and MBNL1 mutually destabilize mRNA. Precise temporal regulation of CUGBP1 and MBNL1 are likely to be essential for accurate control of destabilization of a broad spectrum of genes as well as of alternative splicing events in cell differentiation and tissue development.
Project description:Myotonic dystrophy type 1 (DM1) is an RNA dominant disease in which mutant transcripts containing an expanded CUG repeat (CUGexp) cause muscle dysfunction by interfering with biogenesis of other mRNAs. The toxic effects of mutant RNA are mediated partly through sequestration of splicing regulator Muscleblind-like 1 (Mbnl1), a protein that binds to CUGexp RNA. A gene that is prominently affected encodes chloride channel 1 (Clcn1), resulting in hyperexcitability of muscle (myotonia). To identify DM1-affected genes and study mechanisms for dysregulation, we performed global mRNA profiling in transgenic mice that express CUGexp RNA, as compared to Mbnl1 knockout and Clcn1 null mice. We found that the majority of changes induced by CUGexp RNA in skeletal muscle can be explained by reduced activity of Mbnl1, including many changes that are secondary to myotonia. The pathway most affected comprises genes involved in calcium signaling and homeostasis. Some effects of CUGexp RNA on gene expression are caused by abnormal alternative splicing or downregulation of Mbnl1-interacting mRNAs. However, several of the most highly dysregulated genes showed altered transcription, as indicated by parallel changes of the corresponding premRNAs. These results support the idea that trans-dominant effects of CUGexp RNA on gene expression in this transgenic model may occur at the level of transcription, RNA processing, and mRNA decay, and are mediated mainly but not entirely through sequestration of Mbnl1. Experiment Overall Design: All experiments involved generating expression profiles of quadriceps muscles taken from mice. Experiments 1 and 2: samples were hybridized to Moe430A and Moe430B arrays. Experiment 3: samples were hybridized to Mouse Genome 430 2.0 array. Experiment 1 compared expression profiles of wild-type mice (FVB strain) with two lines, designated 20b and 41, of CUGexp transgenic mice with FVB background. Experiment 2 compared expression profiles of Clcn1-null (myotonic) mice with the wild-type background strain (BALB). Experiment 3 compared expression profiles of Mbnl1-null mice with the wild-type background strain (FVB).
Project description:This SuperSeries is composed of the following subset Series: GSE21233: Expression data from C2C12 mouse myoblast with treatment actinomycin D GSE21235: mRNA immunoprecipitated with CUGBP1 in C2C12 Refer to individual Series
Project description:The Muscleblind-like (Mbnl) family of RNA-binding proteins plays important roles in muscle and eye development and in Myotonic Dystrophy (DM), where expanded CUG or CCUG repeats functionally deplete Mbnl proteins. We identified transcriptome-wide functional and biophysical targets of Mbnl proteins in brain, heart, muscle, and myoblasts using RNA sequencing and crosslinking/immunoprecipitation-sequencing approaches. This analysis identified several hundred splicing events whose regulation depended on Mbnl function, in a pattern indicative of functional interchangeability between Mbnl1 and Mbnl2. A nucleotide resolution RNA map associated repression or activation of exon splicing with Mbnl binding near either 3' splice site or near the downstream 5' splice site, respectively. Transcriptomic analysis of sub-cellular compartments uncovered a global role for Mbnls in regulating localization of mRNAs encoding membrane, synaptic and other proteins in both mouse and Drosophila cells, and Mbnls also contribute to protein secretion. These findings hold several new implications for DM pathogenesis. To assess global functions of Muscleblind proteins, RNA-Seq was performed using WT and Mbnl1 KO brain, heart, and muscle (5 mice each). Additionally, C2C12 mouse myoblasts were depleted of Mbnl1, Mbnl2, or both. Subcellular fractionation experiments were performed to analyze mRNA localization following depletion of Mbnl1 and Mbnl2 in C2C12 mouse myoblasts, and following depletion of Mbnl in Drosophila S-2R+ cells. CLIP-Seq was also performed against Mbnl1 in mouse brain, heart, muscle, and C2C12 myoblasts. Finally, ribosome footprinting was performed with C2C12 mouse myoblasts that were depleted of Mbnl1, Mbnl2, or both.
Project description:Mapping MBNL-regulated genome-wide alternative polyadenylation: We report that depletion of Mbnl proteins in mouse embryo fibroblasts (MEFs), DM mouse model quadriceps muscle, and DM-autopsy muscle tissue leads to mis-regulation of alternative polyadenylation We compared WT, Mbnl1/2KO, Mbnl1/2KO/3siRNA, and Mbnl1/2KO/scrambled siRNA MEFs (n=2 for each group) to evaluate alternative polyadenylation shifts that occur due to progressive loss of Mbnl proteins. We also compared WT (1 day old, and 4 months old, n=2 each) and HSALR mouse model (4 months old, n=2) of myotonic dystrophy for developmental alternative polyadenylation defects in myotonic dystrophy. Finally, we compared control and DM1 autopsy muscle tissues (n=3) for changes in alternative polyadenylation. We performed HITS-CLIP analysis of binding sites of Mbnl1, Mbnl2 and Mbnl3 in MEFs (n=3 each). We also performed HITS-CLIP analysis for major skeletal muscle Mbnl protein, Mbnl1 in FVB WT adult muscle (4 months, n=3). Finally we performed HITS-CLIP analysis for CPSF6 in WT and Mbnl1/2 KO MEFs (n=3 each) Please note that the 'readme_Table.txt' describes the contents of 'Table S*.xlsx' files, and the readme_method.txt include additional details about experiemenal procedures.
Project description:Dramatic changes in gene expression occur in response to extracellular stimuli and during differentiation. Although transcriptional effects are important, alterations in mRNA decay also play a major role in achieving rapid and massive changes in mRNA abundance. Moreover, just as transcription factor activity varies between different cell types, the factors influencing mRNA decay are also cell-type specific.GREs are recognized by CUGBP1, an RNA-binding protein and instability factor whose function is affected in several neuromuscular diseases. To dectect the mRNA associated with CUGBP1, we utilized RNA immunoprecipitation followed by microarray (RIP-Chip) to identify CUGBP1-associated transcripts. To identify the mRNA associated with CUGBP1, we performed the immunoprecipitation followed by microarray (RIP-Chip)
Project description:Myotonic dystrophy (DM) is a multi-systemic disease that severely impacts cardiac and skeletal muscle functions as well as the central nervous system. DM is unusual because it is RNA-mediated disease due to the expression of C(C)UG expansion RNAs that inhibit the activities of the muscleblind-like (MBNL) proteins. In mice, studies using Mbnl1 and Mbnl2 single knockouts have revealed that Mbnl1 plays a predominant role in skeletal and heart muscle alternative splicing regulation while Mbnl2 performs an analogous splicing function in the brain. However, Mbnl single knockout models fail to recapitulate the full-range of adult-onset DM muscle symptoms. Here, we report that Mbnl1; Mbnl2 double knockouts are embryonic lethal while Mbnl1-/-; Mbnl2+/- mice, which express no Mbnl1 and reduced levels of Mbnl2, are viable but develop cardinal features of adult-onset DM cardiac and skeletal muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in both Mbnl1-/- and Mbnl1-/-; Mbnl2+/- knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the MBNL loss-of-function model for DM and provide novel Mbnl compound knockout models to investigate the molecular pathways disrupted by RNA-mediated disease. Mbnl2 protein-RNA interactions were assessed in 4-month-old WT and Mbnl1-/- quadriceps muscles in triplicates by HITS-CLIP.