The Experts below are selected from a list of 1368 Experts worldwide ranked by ideXlab platform
Maurice S. Swanson - One of the best experts on this subject based on the ideXlab platform.
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MBNL1-mediated regulation of differentiation RNAs promotes myofibroblast transformation and the fibrotic response
Nature communications, 2015Co-Authors: Jennifer Davis, Maurice S. Swanson, Apoorva Mohan, Nathan Salomonis, Natasha Ghearing, Suh-chin J. Lin, Jennifer Q. Kwong, Jeffery D. MolkentinAbstract:The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodelling, although the molecular programme underlying this process remains poorly understood. Here we perform a genome-wide screen for genes that control myofibroblast transformation, and identify the RNA-binding protein muscleblind-like1 (MBNL1). MBNL1 overexpression promotes transformation of fibroblasts into myofibroblasts, whereas loss of MBNL1 abrogates transformation and impairs the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Mechanistically, MBNL1 directly binds to and regulates a network of differentiation-specific and cytoskeletal/matrix-assembly transcripts to promote myofibroblast differentiation. One of these transcripts is the nodal transcriptional regulator serum response factor (SRF), whereas another is calcineurin Aβ. CRISPR-Cas9-mediated gene-editing of the MBNL1-binding site within the Srf 3'UTR impairs myofibroblast differentiation, whereas in vivo deletion of Srf in fibroblasts impairs wound healing and fibrosis. These data establish a new RNA-dependent paradigm for myofibroblast formation through MBNL1.
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Muscleblind-like 1 is required for normal heart valve development in vivo
BMC developmental biology, 2015Co-Authors: Ryan J. Coram, Maurice S. Swanson, Samantha J. Stillwagon, Anuradha Guggilam, Michael W. Jenkins, Andrea N. LaddAbstract:Development of the valves and septa of the heart depends on the formation and remodeling of the endocardial cushions in the atrioventricular canal and outflow tract. These cushions are populated by mesenchyme produced from the endocardium by epithelial-mesenchymal transition (EMT). The endocardial cushions are remodeled into the valves at post-EMT stages via differentiation of the mesenchyme and changes in the extracellular matrix (ECM). Transforming growth factor β (TGFβ) signaling has been implicated in both the induction of EMT in the endocardial cushions and the remodeling of the valves at post-EMT stages. We previously identified the RNA binding protein muscleblind-like 1 (MBNL1) as a negative regulator of TGFβ signaling and EMT in chicken endocardial cushions ex vivo. Here, we investigate the role of MBNL1 in endocardial cushion development and valvulogenesis in MBNL1 ∆E3/∆E3 mice, which are null for MBNL1 protein. Collagen gel invasion assays, histology, immunohistochemistry, real-time RT-PCR, optical coherence tomography, and echocardiography were used to evaluate EMT and TGFβ signaling in the endocardial cushions, and morphogenesis, ECM composition, and function of the heart valves. As in chicken, the loss of MBNL1 promotes precocious TGFβ signaling and EMT in the endocardial cushions. Surprisingly, this does not lead to the production of excess mesenchyme, but later valve morphogenesis is aberrant. Adult MBNL1 ∆E3/∆E3 mice exhibit valve dysmorphia with elevated TGFβ signaling, changes in ECM composition, and increased pigmentation. This is accompanied by a high incidence of regurgitation across both inflow and outflow valves. MBNL1 ∆E3/∆E3 mice also have a high incidence of ostium secundum septal defects accompanied by atrial communication, but do not develop overt cardiomyopathy. Together, these data indicate that MBNL1 plays a conserved role in negatively regulating TGFβ signaling, and is required for normal valve morphogenesis and homeostasis in vivo.
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Abstract 12192: MBNL1 Directly Regulates the Alternative Splicing of mRNAs That Promote Myofibroblast Differentiation
Circulation, 2014Co-Authors: Jennifer S. Davis, Maurice S. Swanson, Michelle A. Sargent, Jianjian Shi, Lei Wei, Jeffery D. MolkentinAbstract:Rationale: During the cardiac injury response fibroblasts differentiate into myofibroblasts, a cell type that enhances extracellular matrix production and facilitates ventricular remodeling. To better understand the molecular mechanisms whereby myofibroblasts are generated in the heart we performed a genome-wide screen with 18,000 cDNAs, which identified the RNA-binding protein muscleblind-like splicing regulator 1 (MBNL1), suggesting a novel association between mRNA alternative splicing and the regulation of myofibroblast differentiation. Objective: To determine the mechanism whereby MBNL1 regulates myofibroblast differentiation and the cardiac fibrotic response. Methods and Results: Confirming the results from our genome wide screen, adenoviral-mediated overexpression of MBNL1 promoted transformation of rat cardiac fibroblasts and mouse embryonic fibroblasts (MEFs) into myofibroblasts, similar to the level of conversion obtained by the profibrotic agonist transforming growth factor β (TGFβ). Antithetically, MBNL1 -/- MEFs were refractory to TGFβ-induced myofibroblast differentiation. MBNL1 expression is induced in transforming fibroblasts in response to TGFβ and angiotensin II. These results were extended in vivo by analysis of dermal wound healing, a process dependent on myofibroblast differentiation and their proper activity. By day 6 control mice had achieved 82% skin wound closure compared with only 40% in MBNL1 -/- mice. Moreover, MBNL1 -/- mice had reduced survival following myocardial infarction injury due to defective fibrotic scar formation and healing. High throughput RNA sequencing (RNAseq) and RNA immunoprecipitation revealed that MBNL1 directly regulates the alternative splicing of transcripts for myofibroblast signaling factors and cytoskeletal-assembly elements. Functional analysis of these factors as mediators of MBNL1 activity is also described here. Conclusions: Collectively, our data suggest that MBNL1 coordinates myofibroblast transformation by directly mediating the alternative splicing of an array of mRNAs encoding differentiation-specific signaling transcripts, which then alter the fibroblast proteome for myofibroblast structure and function.
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Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy
Nature Structural & Molecular Biology, 2010Co-Authors: Hongqing Du, Maurice S. Swanson, Melissa S Cline, Daniel L Tuttle, Tyson A Clark, John Paul Donohue, Megan P Hall, Lily Shiue, Robert J Osborne, Charles A. ThorntonAbstract:Myotonic dystrophy is a CUG repeat expansion disease and mice deficient in MBNL1, an RNA binding protein, show many characteristics of the disease. Comparison of gene expression profiles of two mouse models of the disease reveals that CUG repeat expansions have two effects – loss of MBNL1 function that leads to altered splicing and loss of an as yet unknown function that disrupts extracellular matrix protein mRNA regulation. The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG^exp RNA). To test whether CUG^exp RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG^exp transgene and another homozygous for a defective muscleblind 1 ( MBNL1 ) gene. Strong correlation in splicing changes for ∼100 new MBNL1-regulated exons indicates that loss of MBNL1 explains >80% of the splicing pathology due to CUG^exp RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of MBNL1, indicating that CUG^exp RNA has MBNL1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG^exp RNA causes two separate effects: loss of MBNL1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.
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aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy
Nature Structural & Molecular Biology, 2010Co-Authors: Melissa S Cline, Charles A. Thornton, Maurice S. Swanson, Robert Osborne, Daniel L Tuttle, Tyson A Clark, John Paul Donohue, Megan P Hall, Lily Shiue, Manuel AresAbstract:The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (MBNL1) gene. Strong correlation in splicing changes for approximately 100 new MBNL1-regulated exons indicates that loss of MBNL1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of MBNL1, indicating that CUG(exp) RNA has MBNL1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of MBNL1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.
J. Andrew Berglund - One of the best experts on this subject based on the ideXlab platform.
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Pseudouridine Modification Inhibits Muscleblind-like 1 (MBNL1) Binding to CCUG Repeats and Minimally Structured RNA through Reduced RNA Flexibility.
The Journal of biological chemistry, 2017Co-Authors: Elaine Delorimier, Melissa N. Hinman, Jeremy Copperman, Kausiki Datta, Marina Guenza, J. Andrew BerglundAbstract:Abstract Myotonic dystrophy type 2 is a genetic neuromuscular disease caused by the expression of expanded CCUG repeat RNAs from the non-coding region of the CCHC-type zinc finger nucleic acid-binding protein (CNBP) gene. These CCUG repeats bind and sequester a family of RNA-binding proteins known as Muscleblind-like 1, 2, and 3 (MBNL1, MBNL2, and MBNL3), and sequestration plays a significant role in pathogenicity. MBNL proteins are alternative splicing regulators that bind to the consensus RNA sequence YGCY (Y = pyrimidine). This consensus sequence is found in the toxic RNAs (CCUG repeats) and in cellular RNA substrates that MBNL proteins have been shown to bind. Replacing the uridine in CCUG repeats with pseudouridine (Ψ) resulted in a modest reduction of MBNL1 binding. Interestingly, Ψ modification of a minimally structured RNA containing YGCY motifs resulted in more robust inhibition of MBNL1 binding. The different levels of inhibition between CCUG repeat and minimally structured RNA binding appear to be due to the ability to modify both pyrimidines in the YGCY motif, which is not possible in the CCUG repeats. Molecular dynamic studies of unmodified and pseudouridylated minimally structured RNAs suggest that reducing the flexibility of the minimally structured RNA leads to reduced binding by MBNL1.
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Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy.
PLoS genetics, 2016Co-Authors: Stacey D. Wagner, Charles A. Thornton, Adam J. Struck, Riti Gupta, Dylan R. Farnsworth, Amy E. Mahady, Katy Eichinger, Eric T. Wang, J. Andrew BerglundAbstract:Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.
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Autoregulated splicing of muscleblind-like 1 (MBNL1) Pre-mRNA.
The Journal of biological chemistry, 2011Co-Authors: Devika P. Gates, Leslie A. Coonrod, J. Andrew BerglundAbstract:Muscleblind-like 1 (MBNL1) is a splicing factor whose improper cellular localization is a central component of myotonic dystrophy. In myotonic dystrophy, the lack of properly localized MBNL1 leads to missplicing of many pre-mRNAs. One of these events is the aberrant inclusion of exon 5 within the MBNL1 pre-mRNA. The region of the MBNL1 gene that includes exon 5 and flanking intronic sequence is highly conserved in vertebrate genomes. The 3′-end of intron 4 is non-canonical in that it contains a predicted branch point that is 141 nucleotides from the 3′-splice site and an AAG 3′-splice site. Using a minigene that includes exon 4, intron 4, exon 5, intron 5, and exon 6 of MBNL1, we showed that MBNL1 regulates inclusion of exon 5. Mapping of the intron 4 branch point confirmed that branching occurs primarily at the predicted distant branch point. Structure probing and footprinting revealed that the highly conserved region between the branch point and 3′-splice site is primarily unstructured and that MBNL1 binds within this region of the pre-mRNA. Deletion of the MBNL1 response element eliminated MBNL1 splicing regulation and led to complete inclusion of exon 5, which is consistent with the suppressive effect of MBNL1 on splicing.
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The four Zn fingers of MBNL1 provide a flexible platform for recognition of its RNA binding elements
BMC molecular biology, 2011Co-Authors: Danielle M. Cass, Devika P. Gates, Rachel Hotchko, Paul R. Barber, Kimberly Jones, J. Andrew BerglundAbstract:Background Muscleblind-like 1 (MBNL1) is an alternative splicing factor containing four CCCH Zinc fingers (ZnFs). The sequestration of MBNL1 by expanded CUG and CCUG repeats is a major component in causing myotonic dystrophy. In addition to binding the structured expanded CUG and CCUG repeats; previous results suggested that MBNL1 binds single-stranded RNAs containing GC dinucleotides.
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MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing
Nucleic acids research, 2010Co-Authors: Emily S. Goers, Devika P. Gates, Jamie Purcell, Rodger B. Voelker, J. Andrew BerglundAbstract:Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs.
Charles A. Thornton - One of the best experts on this subject based on the ideXlab platform.
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Dose-Dependent Regulation of Alternative Splicing by MBNL Proteins Reveals Biomarkers for Myotonic Dystrophy.
PLoS genetics, 2016Co-Authors: Stacey D. Wagner, Charles A. Thornton, Adam J. Struck, Riti Gupta, Dylan R. Farnsworth, Amy E. Mahady, Katy Eichinger, Eric T. Wang, J. Andrew BerglundAbstract:Alternative splicing is a regulated process that results in expression of specific mRNA and protein isoforms. Alternative splicing factors determine the relative abundance of each isoform. Here we focus on MBNL1, a splicing factor misregulated in the disease myotonic dystrophy. By altering the concentration of MBNL1 in cells across a broad dynamic range, we show that different splicing events require different amounts of MBNL1 for half-maximal response, and respond more or less steeply to MBNL1. Motifs around MBNL1 exon 5 were studied to assess how cis-elements mediate the MBNL1 dose-dependent splicing response. A framework was developed to estimate MBNL concentration using splicing responses alone, validated in the cell-based model, and applied to myotonic dystrophy patient muscle. Using this framework, we evaluated the ability of individual and combinations of splicing events to predict functional MBNL concentration in human biopsies, as well as their performance as biomarkers to assay mild, moderate, and severe cases of DM.
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Two high-throughput screening assays for aberrant RNA-protein interactions in myotonic dystrophy type 1.
Analytical and bioanalytical chemistry, 2012Co-Authors: Catherine Z. Chen, Charles A. Thornton, Krzysztof Sobczak, Jason Hoskins, Noel Southall, Juan J. Marugan, Wei Zheng, Christopher P. AustinAbstract:Myotonic dystrophy type 1 (DM1), the most prevalent form of adult muscular dystrophy, is caused by expansion of a CTG repeat in the 3′ untranslated region of the DM protein kinase (DMPK) gene. The pathogenic effects of the CTG expansion arise from the deleterious effects of the mutant transcript. RNA with expanded CUG tracts alters the activities of several RNA binding proteins, including muscleblind-like 1 (MBNL1). MBNL1 becomes sequestered in nuclear foci in complex with the expanded CUG-repeat RNA. The resulting loss of MBNL1 activity causes misregulated alternative splicing of multiple genes, leading to symptoms of DM1. The binding interaction between MBNL1 and mutant RNA could be a key step in the pathogenesis of DM1 and serves as a potential target for therapeutic intervention. We have developed two high-throughput screens suitable assays using both homogenous time-resolved fluorescence energy transfer and AlphaScreen technologies to detect the binding of a C-terminally His-tagged MBNL1 and a biotinylated (CUG)12 RNA. These assays are homogenous and successfully miniaturized to 1,536-well plate format. Both assays were validated and show robust signal-to-basal ratios and Z′ factors.
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aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy
Nature Structural & Molecular Biology, 2010Co-Authors: Melissa S Cline, Charles A. Thornton, Maurice S. Swanson, Robert Osborne, Daniel L Tuttle, Tyson A Clark, John Paul Donohue, Megan P Hall, Lily Shiue, Manuel AresAbstract:The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG(exp) RNA). To test whether CUG(exp) RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG(exp) transgene and another homozygous for a defective muscleblind 1 (MBNL1) gene. Strong correlation in splicing changes for approximately 100 new MBNL1-regulated exons indicates that loss of MBNL1 explains >80% of the splicing pathology due to CUG(exp) RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of MBNL1, indicating that CUG(exp) RNA has MBNL1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG(exp) RNA causes two separate effects: loss of MBNL1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.
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Aberrant alternative splicing and extracellular matrix gene expression in mouse models of myotonic dystrophy
Nature Structural & Molecular Biology, 2010Co-Authors: Hongqing Du, Maurice S. Swanson, Melissa S Cline, Daniel L Tuttle, Tyson A Clark, John Paul Donohue, Megan P Hall, Lily Shiue, Robert J Osborne, Charles A. ThorntonAbstract:Myotonic dystrophy is a CUG repeat expansion disease and mice deficient in MBNL1, an RNA binding protein, show many characteristics of the disease. Comparison of gene expression profiles of two mouse models of the disease reveals that CUG repeat expansions have two effects – loss of MBNL1 function that leads to altered splicing and loss of an as yet unknown function that disrupts extracellular matrix protein mRNA regulation. The common form of myotonic dystrophy (DM1) is associated with the expression of expanded CTG DNA repeats as RNA (CUG^exp RNA). To test whether CUG^exp RNA creates a global splicing defect, we compared the skeletal muscle of two mouse models of DM1, one expressing a CTG^exp transgene and another homozygous for a defective muscleblind 1 ( MBNL1 ) gene. Strong correlation in splicing changes for ∼100 new MBNL1-regulated exons indicates that loss of MBNL1 explains >80% of the splicing pathology due to CUG^exp RNA. In contrast, only about half of mRNA-level changes can be attributed to loss of MBNL1, indicating that CUG^exp RNA has MBNL1-independent effects, particularly on mRNAs for extracellular matrix proteins. We propose that CUG^exp RNA causes two separate effects: loss of MBNL1 function (disrupting splicing) and loss of another function that disrupts extracellular matrix mRNA regulation, possibly mediated by Mbnl2. These findings reveal unanticipated similarities between DM1 and other muscular dystrophies.
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Pentamidine reverses the splicing defects associated with myotonic dystrophy
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: M. Bryan Warf, Charles A. Thornton, Masayuki Nakamori, Catherine M. Matthys, J. Andrew BerglundAbstract:Myotonic dystrophy (DM) is a genetic disorder caused by the expression (as RNA) of expanded CTG or CCTG repeats. The alternative splicing factor MBNL1 is sequestered to the expanded RNA repeats, resulting in missplicing of a subset of pre-mRNAs linked to symptoms found in DM patients. Current data suggest that if MBNL1 is released from sequestration, disease symptoms may be alleviated. We identified the small molecules pentamidine and neomycin B as compounds that disrupt MBNL1 binding to CUG repeats in vitro. We show in cell culture that pentamidine was able to reverse the missplicing of 2 pre-mRNAs affected in DM, whereas neomycin B had no effect. Pentamidine also significantly reduced the formation of ribonuclear foci in tissue culture cells, releasing MBNL1 from the foci in the treated cells. Furthermore, pentamidine partially rescued splicing defects of 2 pre-mRNAs in mice expressing expanded CUG repeats.
Thomas A. Cooper - One of the best experts on this subject based on the ideXlab platform.
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Muscleblind-like 1 activates insulin receptor exon 11 inclusion by enhancing U2AF65 binding and splicing of the upstream intron
Nucleic acids research, 2013Co-Authors: Gloria V. Echeverria, Thomas A. CooperAbstract:Alternative splicing regulates developmentally and tissue-specific gene expression programs, disruption of which have been implicated in numerous diseases. Muscleblind-like 1 (MBNL1) regulates splicing transitions, which are disrupted on loss of MBNL1 function in myotonic dystrophy type 1 (DM1). One such event is MBNL1-mediated activation of insulin receptor exon 11 inclusion, which requires an intronic enhancer element downstream of exon 11. The mechanism of MBNL1-mediated activation of exon inclusion is unknown. We developed an in vitro splicing assay, which robustly recapitulates MBNL1-mediated splicing activation of insulin receptor exon 11 and found that MBNL1 activates removal of the intron upstream of exon 11 upon binding its functional response element in the downstream intron. MBNL1 enhances early spliceosome assembly as evidenced by enhanced complex A formation and binding of U2 small nuclear ribonucleoprotein auxiliary factor 65 kDa subunit (U2AF65) on the upstream intron. We demonstrated that neither the 5′ splice site nor exon 11 sequences are required for MBNL1-activated U2AF65 binding. Interestingly, the 5′ splice site is required for MBNL1-mediated activation of upstream intron removal, although MBNL1 has no effect on U1 snRNA recruitment. These results suggest that MBNL1 directly activates binding of U2AF65 to enhance upstream intron removal to ultimately activate alternative exon inclusion.
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Identification of MBNL1 and MBNL3 domains required for splicing activation and repression
Nucleic acids research, 2010Co-Authors: Ioannis Grammatikakis, Young-hwa Goo, Gloria V. Echeverria, Thomas A. CooperAbstract:Muscleblind-like 1 (MBNL1) is a splicing regulator that controls developmentally regulated alternative splicing of a large number of exons including exon 11 of the Insulin Receptor (IR) gene and exon 5 of the cardiac Troponin T (cTNT) gene. There are three paralogs of MBNL in humans, all of which promote IR exon 11 inclusion and cTNT exon 5 skipping. Here, we identify a cluster of three binding sequences located downstream of IR exon 11 that constitute the MBNL1 response element and a weaker response element in the upstream intron. In addition, we used sequential deletions to define the functional domains of MBNL1 and MBNL3. We demonstrate that the regions required for splicing regulation are separate from the two pairs of zinc-finger RNA-binding domains. MBNL1 and MBNL3 contain core regulatory regions for both activation and repression located within an 80-amino-acid segment located downstream of the N-terminal zinc-finger pair. Deletions of these regions abolished regulation without preventing RNA binding. These domains have common features with the CUG-BP and ETR3-like Factor (CELF) family of splicing regulators. These results have identified protein domains required for splicing repression and activation and provide insight into the mechanism of splicing regulation by MBNL proteins.
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Expanded CTG repeats within the DMPK 3' UTR causes severe skeletal muscle wasting in an inducible mouse model for myotonic dystrophy.
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: James P Orengo, Pierre Chambon, Daniel Metzger, Dennis R Mosier, G Jackson Snipes, Thomas A. CooperAbstract:Severe skeletal muscle wasting is the most debilitating symptom experienced by individuals with myotonic dystrophy type 1 (DM1). We present a DM1 mouse model with inducible and skeletal muscle-specific expression of large tracts of CTG repeats in the context of DMPK exon 15. These mice recapitulate many findings associated with DM1 skeletal muscle, such as CUG RNA foci with Muscleblind-like 1 (MBNL1) protein colocalization, misregulation of developmentally regulated alternative splicing events, myotonia, characteristic histological abnormalities, and increased CUGBP1 protein levels. Importantly, this DM1 mouse model recapitulates severe muscle wasting, which has not been reported in models in which depletion of MBNL1 is the main feature. Using these mice, we discovered previously undescribed alternative splicing events that are responsive to CUGBP1 and not MBNL, and these events were found to be misregulated in individuals with DM1. Our results indicate that increased CUGBP1 protein levels are associated with DMPK-CUG RNA expression, suggesting a role for CUGBP1-specific splicing or cytoplasmic functions in muscle wasting.
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MBNL1 and CUGBP1 modify expanded CUG-induced toxicity in a Drosophila model of myotonic dystrophy type 1.
Human molecular genetics, 2006Co-Authors: Maria De Haro, Thomas A. Cooper, Ismael Al-ramahi, Beatrice De Gouyon, Lubna Ukani, Alberto L. Rosa, Nuno André Faustino, Tetsuo Ashizawa, Juan BotasAbstract:Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG expansion in the 3 0 UTR of the dystrophia myotonica protein kinase (DMPK) gene. It has been hypothesized that the pathogenesis in DM1 is triggered by a toxic gain of function of the expanded DMPK RNA. This expanded RNA is retained in nuclear foci where it sequesters and induces alterations in the levels of RNA-binding proteins (RNA-BP). To model DM1 and study the implication of RNA-BP in CUG-induced toxicity, we have generated a Drosophila DM1 model expressing a non-coding mRNA containing 480 interrupted CUG repeats; i.e. [(CUG)20CUCGA]24 .T his (iCUG)480 transcript accumulates in nuclear foci and its expression leads to muscle wasting and degeneration in Drosophila. We also report that altering the levels of two RNA-BP known to be involved in DM1 pathogenesis, MBNL1 and CUGBP1, modify the (iCUG)480 degenerative phenotypes. Expanded CUG-induced toxicity in Drosophila is suppressed when MBNL1 expression levels are increased, and enhanced when MBNL1 levels are reduced. In addition, (iCUG)480 also causes a decrease in the levels of soluble MBNL1 that is sequestered in the CUG-containing nuclear foci. In contrast, increasing the levels of CUGBP1 worsens (iCUG)480-induced degeneration even though CUGBP1 distribution is not altered by the expression of the expanded triplet repeat. Our data supports a mechanism for DM1 pathogenesis in which decreased levels of MBNL and increased levels of CUGBP mediate the RNA-induced toxicity observed in DM1. Perhaps more importantly, they also provide proof of the principle that CUG-induced muscle toxicity can be suppressed.
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colocalization of muscleblind with rna foci is separable from mis regulation of alternative splicing in myotonic dystrophy
Journal of Cell Science, 2005Co-Authors: Thai H Ho, Maurice S. Swanson, Rajesh S Savkur, Michael G Poulos, Michael A Mancini, Thomas A. CooperAbstract:Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing mis-regulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.
John W. Day - One of the best experts on this subject based on the ideXlab platform.
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rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences
Nature Communications, 2018Co-Authors: Chantal Sellier, Estefanía Cerro-herreros, Markus Blatter, Fernande Freyermuth, Angeline Gaucherot, Frank Ruffenach, Partha Sarkar, Jack Puymirat, Bjarne Udd, John W. DayAbstract:Myotonic dystrophy (DM) type 2 is a neuromuscular pathology caused by large expansions of CCTG repeats. Here the authors find that rbFOX1 RNA binding protein binds to CCUG RNA repeats and competes with MBNL1 for the binding to CCUG repeats, releasing MBNL1 from sequestration in DM2 muscle cells. Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.
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rbFOX1/MBNL1 competition for CCUG RNA repeats binding contributes to myotonic dystrophy type 1/type 2 differences
Nature communications, 2018Co-Authors: Chantal Sellier, Estefanía Cerro-herreros, Markus Blatter, Fernande Freyermuth, Angeline Gaucherot, Frank Ruffenach, Jack Puymirat, Bjarne Udd, Partha S. Sarkar, John W. DayAbstract:Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.
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rbfox1 MBNL1 competition for ccug rna repeats binding contributes to myotonic dystrophy type 1 type 2 differences
Nature Communications, 2018Co-Authors: Chantal Sellier, Markus Blatter, Fernande Freyermuth, Angeline Gaucherot, Frank Ruffenach, Jack Puymirat, Bjarne Udd, Partha S. Sarkar, Estefania Cerroherreros, John W. DayAbstract:Myotonic dystrophy type 1 and type 2 (DM1, DM2) are caused by expansions of CTG and CCTG repeats, respectively. RNAs containing expanded CUG or CCUG repeats interfere with the metabolism of other RNAs through titration of the Muscleblind-like (MBNL) RNA binding proteins. DM2 follows a more favorable clinical course than DM1, suggesting that specific modifiers may modulate DM severity. Here, we report that the rbFOX1 RNA binding protein binds to expanded CCUG RNA repeats, but not to expanded CUG RNA repeats. Interestingly, rbFOX1 competes with MBNL1 for binding to CCUG expanded repeats and overexpression of rbFOX1 partly releases MBNL1 from sequestration within CCUG RNA foci in DM2 muscle cells. Furthermore, expression of rbFOX1 corrects alternative splicing alterations and rescues muscle atrophy, climbing and flying defects caused by expression of expanded CCUG repeats in a Drosophila model of DM2.