Project description:mbnl knockout Danio rerio were created using CRISPR-Cas9, including single mbnl paralog knockouts, double mbnl paralog knockouts, and a triple mbnl paralog knockout. RNA-Seq was performed using skeletal muscle of three biological replicates of four month old fish.
Project description:MBNL1 is a known splicing factor and is related to Myotonic Dystrophy (DM). This study examines the tissue specific splicing patterns of MBNL1 using a mutant and wild type mouse across three tissues (heart,brain,quadricep) related publications: Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy. Du H, etal Nat Struct Mol Biol. 2010 Feb;17(2):187-93. and Hum Mol Genet. 2006 Jul 1;15(13):2087-97. Failure of MBNL1-dependent post-natal splicing transitions in myotonic dystrophy. Lin X, Miller JW, Mankodi A, Kanadia RN, Yuan Y, Moxley RT, Swanson MS, Thornton CA. We examined quadricep,heart and brain of a mouse MBNL1 mutant to test whether MBNL mutants creates a tissue specific splicing defect. These samples were compared to the tissues of a wild type mouse.
Project description:Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete. In the RNAcompete assay, a purified GST-tagged protein is incubated with an excess of RNA pool and bound RNA from individual pulldown experiments are directly labeled and hybridized to a custom Agilent 244K microarray.
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
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:Here, we analyze the RNA-binding preferences of the planarian smed-mbnl-like-2, smed-mbnl-1 (isoform X1), smed-bruli, smed-mbnl-1 (isoform Xins), and smed-mbnl-like-1 protein using RNAcompete.
Project description:The study compares gene expression profile at several stages post amputation of the adult zebrafish ventricular heart between zebrafish mutants and WT siblings. The first experiment was to identify genes that are activated in response to cardiac injury at 3 and 7 days post amputation (dpa). Dusp6 mutant hearts were reported to show an enhanced regenerative response. For this experiment, bulk RNA seq was obtained from WT and Dusp6 mutant hearts and genes increased at 3 and 7 dpa were identified. The forkhead transcription factor, foxm1, showed increased expression in cardiomyocytes and follow up studies show that it is required to regulate cardiomyocyte proliferation. This was further explored with RNA-seq experiments comparing WT and foxm1 mutant hearts at 3dpa. We identified genes normally expressed in proliferating cells to be decreased in the foxm1 mutants.
Project description:The mRNA cap-binding protein eIF4E1b is critical for female germline development in zebrafish. To study the effect of eIF4E1b loss in zebrafish, we isolated gonads with a high expression of ziwi:GFP (female germline marker) from wild-type and eif4e1b mutant juveniles and performed RNA-seq.
Project description:Alternative splicing has emerged as a fundamental mechanism not only for the diversification of protein isoforms but also for the spatiotemporal control of development. Therefore, a better understanding of how this mechanism is regulated has the potential not only to elucidate fundamental biological principles, but also to decipher pathological mechanisms implicated in diseases where normal splicing networks are misregulated. Here, we took advantage of human pluripotent stem cells to decipher during human myogenesis the role of MBNL proteins, a family of tissue-specific splicing regulators whose loss of function is associated with Myotonic Dystrophy type 1, an inherited neuromuscular disease. Thanks to the CRISPR/Cas9 technology, we generated human-induced pluripotent stem cells (hiPSCs) depleted in MBNL proteins and evaluated the molecular and functional consequences of this loss on the generation of skeletal muscle cells. Our results indicated that MBNL proteins are specifically required for the late myogenic maturation but not for early myogenic commitment. By a transcriptomic analysis, we further demonstrated that MBNL proteins are not only important for the regulation of alternative splicing but also for the regulation of gene expression. Together, our study reveals the temporal requirement of MBNL proteins in human myogenesis and should facilitate the identification of new therapeutic strategies capable to cope the loss of function of these MBNL proteins.