scholarly journals Targeting Toxic Nuclear RNA Foci with CRISPR-Cas13 to Treat Myotonic Dystrophy

2019 ◽  
Author(s):  
Pornthida Poosala ◽  
Sean R. Lindley ◽  
Kelly M. Anderson ◽  
Douglas M. Anderson
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
Live Cells ◽  
Nuclear Retention ◽  
Rna Foci ◽  
Nuclear Rna ◽  
Nuclear Foci ◽  
Dystrophia Myotonica Protein Kinase

Human monogenetic diseases can arise from the aberrant expansion of tandem nucleotide repeat sequences, which when transcribed into RNA, can misfold and aggregate into toxic nuclear foci1. Nuclear retention of repeat-containing RNAs can disrupt their normal expression and induce widespread splicing defects by sequestering essential RNA binding proteins. Among the most prevalent of these disorders is myotonic dystrophy type 1 (DM1), a disease occurring from the expression of a noncoding CTG repeat expansion in the 3’UTR of the human dystrophia myotonica protein kinase (DMPK) gene2,3. Here we show that RNA-binding CRISPR-Cas13, with a robust non-classical nuclear localization signal, can be efficiently targeted to toxic nuclear RNA foci for either visualization or cleavage, tools we named hilightR and eraseR, respectively. HilightR combines catalytically dead Cas13b (dCas13b) with a fluorescent protein to directly visualize CUG repeat RNA foci in the nucleus of live cells, allowing for quantification of foci number and observation of foci dynamics. EraseR utilizes the intrinsic endoribonuclease activity of Cas13b, targeted to nuclear CUG repeat RNA, to disrupt nuclear foci. These studies demonstrate the potential for targeting toxic nuclear RNA foci directly with CRISPR-Cas13 for either the identification or treatment of DM1. The efficient and sequence programmable nature of CRISPR-Cas13 systems will allow for rapid targeting and manipulation of other human nuclear RNA disorders, without the associated risks of genome editing.

scholarly journals RNA Gelation in Repeat Expansion Disorders

2017 ◽  
Author(s):  
Ankur Jain ◽  
Ronald D. Vale
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neuromuscular Disorders ◽  
Cag Repeats ◽  
Live Cells ◽  
Critical Threshold ◽  
Rna Foci ◽  
Repeat Expansions ◽  
Nucleotide Repeats

Expansions of short nucleotide repeats in the protein coding and non-coding regions of >30 genes produce a variety of neurological and neuromuscular disorders including Huntington’s disease (CAG repeats), muscular dystrophy (CTG repeats) and amyotrophic lateral sclerosis (GGGGCC repeats) [1-3]. Expression of expanded repeats alone is sufficient to recapitulate disease pathology in animal models [4-6]. Repeat-containing transcripts accumulate in the nucleus as aberrant “RNA foci” [7-10] and sequester numerous RNA binding proteins [11,12], leading to a disruption of cellular homeostasis [13,14]. Interestingly, RNA foci, as well as the disease symptoms, only manifest at a critical threshold of nucleotide repeats: >30 for CAG/CTG expansions [1] and >7 for the GGGGCC expansion [15]. However, the reason for this characteristic threshold, as well as the molecular mechanism of foci formation, remain unresolved [16]. Here, we show that nucleotide repeat expansions in RNA create templates for multivalent Watson-Crick (CAG/CUG expansions) or Hoogsteen (GGGGCC expansion) base-pairing. These multivalent interactions cause purified RNAs containing repeat expansions to undergo a sol-gel transition and form micron-sized clusters. Reflecting an increase in the valency for intermolecular hybridization, the gelation of purified RNA only occurs above a critical number of trinucleotide or hexanucleotide repeats. These thresholds for in vitro RNA gelation are similar to those associated with manifestation of disease. By visualizing RNA in live cells, we show that nuclear foci form as a result of phase separation of the repeat-containing RNA and that these foci can be dissolved by agents that disrupt RNA gelation in vitro. Analogous to protein aggregation disorders, our results suggest that the sequence-specific gelation of RNAs could be a contributing factor to neurological disease.

scholarly journals Short Tandem Repeat Expansions and RNA-Mediated Pathogenesis in Myotonic Dystrophy

2019 ◽  
Vol 20 (13) ◽  
pp. 3365 ◽  
Author(s):  
Łukasz J. Sznajder ◽  
Maurice S. Swanson
Keyword(s):  
Myotonic Dystrophy ◽  
Tandem Repeat ◽  
Short Tandem Repeat ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Foci ◽  
Repeat Expansions ◽  
Multivalent Interactions ◽  
Short Tandem

Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant DMPK (DM1) and CNBP (DM2) genes results in the synthesis of CUG and CCUG repeat expansion (CUGexp, CCUGexp) RNAs, respectively. These CUGexp and CCUGexp RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.

scholarly journals Pathogenic mechanisms of myotonic dystrophy

2009 ◽  
Vol 37 (6) ◽  
pp. 1281-1286 ◽  
Author(s):  
Johanna E. Lee ◽  
Thomas A. Cooper
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Genetic Disorder ◽  
Mrna Translation ◽  
Loss Of Function ◽  
Downstream Effects ◽  
Expanded Allele ◽  
Dystrophia Myotonica Protein Kinase ◽  
Dm Type 2

DM (myotonic dystrophy) is a dominantly inherited genetic disorder that is the most common cause of muscular dystrophy in adults affecting 1 in 8500 individuals worldwide. Different microsatellite expansions in two loci cause different forms of the disease that share similar features: DM1 (DM type 1) is caused by a tri- (CTG) nucleotide expansion within the DMPK (dystrophia myotonica protein kinase) 3′-untranslated region and DM2 (DM type 2) is caused by a tetra- (CCTG) nucleotide expansion within intron 1 of the ZNF9 (zinc finger 9) gene. The pathogenic mechanism of this disease involves the RNA transcribed from the expanded allele containing long tracts of (CUG)n or (CCUG)n. The RNA results in a toxic effect through two RNA-binding proteins: MBNL1 (muscleblind-like 1) and CUGBP1 (CUG-binding protein 1). In DM1, MBNL1 is sequestered on CUG repeat-containing RNA resulting in its loss-of-function, while CUGBP1 is up-regulated through a signalling pathway. The downstream effects include disrupted regulation of alternative splicing, mRNA translation and mRNA stability, which contribute to the multiple features of DM1. This review will focus on the RNA gain-of-function disease mechanism, the important roles of MBNL1 and CUGBP1 in DM1, and the relevance to other RNA dominant disorders.

scholarly journals Nucleo-cytoplasmic shuttling of splicing factor SRSF1 is required for development and cilia function

2020 ◽  
Author(s):  
Fiona Haward ◽  
Magdalena M. Maslon ◽  
Patricia L. Yeyati ◽  
Nicolas Bellora ◽  
Jan N. Hansen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mouse Model ◽  
Rna Splicing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Small Body ◽  
Splicing Factor ◽  
Nuclear Retention ◽  
Ciliary Ultrastructure ◽  
Retention Signal

AbstractShuttling RNA-binding proteins coordinate nuclear and cytoplasmic steps of gene expression. The SR family proteins regulate RNA splicing in the nucleus and a subset of them, including SRSF1, shuttles between the nucleus and cytoplasm affecting post-splicing processes. However, the physiological significance of this remains unclear. Here, we used genome editing to knock-in a nuclear retention signal (NRS) in Srsf1 to create a mouse model harboring an SRSF1 protein that is retained exclusively in the nucleus. Srsf1NRS/NRS mutants displayed small body size, hydrocephalus and immotile sperm, all traits associated with ciliary defects. We observed reduced translation of a subset of mRNAs and decreased abundance of proteins involved in multiciliogenesis, with disruption of ciliary ultrastructure and motility in cells derived from this mouse model. These results demonstrate that SRSF1 shuttling is used to reprogram gene expression networks in the context of high cellular demands, as observed here, during motile ciliogenesis.

scholarly journals Fluorescence Correlation and Cross-Correlation Spectroscopy Unveil Cytoplasmic mRNP Composition and Dynamics

2020 ◽  
Author(s):  
Àngels Mateu-Regué ◽  
Jan Christiansen ◽  
Christian Hellriegel ◽  
Finn Cilius Nielsen
Keyword(s):  
Life Cycle ◽  
Dynamic Analysis ◽  
Cross Correlation ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Correlation Spectroscopy ◽  
Live Cells ◽  
Macromolecular Complexes ◽  
Fluorescence Correlation ◽  
Macromolecular Composition

ABSTRACTUnderstanding the mRNA life cycle requires analysis of the dynamic macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study mRNP complexes because they readily provide information about the molecular composition, stoichiometry, heterogeneity and dynamics of the particles. We developed FCS protocols for analysis of live cells and cellular lysates, and demonstrate the feasibility of analysing common cytoplasmic mRNPs composed of core factor YBX1, IMPs (or IGF2BPs) and their interactions with other RNA binding proteins such as PABPC1, ELAVL2 (HuB), STAU1 and FMRP. FCCS corroborated previously reported RNA dependent interactions between the factors and provided an estimate of the relative overlap between the factors in the mRNPs. In this way FCS and FCCS provide a new and useful approach for the quantitative and dynamic analysis of mRNP macromolecular complexes that may complement current biochemical approaches.

scholarly journals Brain Pathogenesis and Potential Therapeutic Strategies in Myotonic Dystrophy Type 1

2021 ◽  
Vol 13 ◽  
Author(s):  
Jie Liu ◽  
Zhen-Ni Guo ◽  
Xiu-Li Yan ◽  
Yi Yang ◽  
Shuo Huang
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Brain Diseases ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Multiple Systems ◽  
Intervention Measures ◽  
Therapeutic Tools

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy that affects multiple systems including the muscle and heart. The mutant CTG expansion at the 3′-UTR of the DMPK gene causes the expression of toxic RNA that aggregate as nuclear foci. The foci then interfere with RNA-binding proteins, affecting hundreds of mis-spliced effector genes, leading to aberrant alternative splicing and loss of effector gene product functions, ultimately resulting in systemic disorders. In recent years, increasing clinical, imaging, and pathological evidence have indicated that DM1, though to a lesser extent, could also be recognized as true brain diseases, with more and more researchers dedicating to develop novel therapeutic tools dealing with it. In this review, we summarize the current advances in the pathogenesis and pathology of central nervous system (CNS) deficits in DM1, intervention measures currently being investigated are also highlighted, aiming to promote novel and cutting-edge therapeutic investigations.

Abstract P104: Reactivation of Embryonic Gene Program due to CUG Repeat RNA Expression in Myotonic Dystrophy

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Auinash Kalsotra ◽  
Ravi Singh ◽  
Chad Creighton ◽  
Thomas Cooper
Keyword(s):  
Gene Expression ◽  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Expression Patterns ◽  
Causative Mutation ◽  
Inherited Disease ◽  
Aberrant Expression ◽  
Organ Systems ◽  
General Response

Myotonic dystrophy type 1 (DM1) is a dominantly inherited disease that affects multiple organ systems. Cardiac involvement, which is characterized by conduction defects and arrhythmias, is the second leading cause of death in DM1 patients. The causative mutation is a CTG expansion in the 3' untranslated region of DMPK gene resulting in aberrant expression of CUG repeat RNA that accumulates into nuclear foci and causes misregulation in alternative splicing. Here we show that heart-specific and inducible expression of CUG repeat RNA in a DM1 mouse model results in global reactivation of embryonic gene expression program in adult heart that is distinct from a general hypertrophic stress response. Using q-PCR TaqMan arrays, we identified 54 miRNAs that were differentially expressed in DM1 mouse hearts one week following induction of CUG repeat RNA. Interestingly, 83% (45/54) of them exhibited a developmental shift in expression towards the embryonic pattern. Because over 90% (41/45) of them were down regulated within 72 hr after induction of repeat RNA and only 2/22 examined decreased in two unrelated mouse models of heart disease, we conclude their reduced expression is specific to DM1 and not simply a general response to cardiac injury. Microarray studies revealed a developmental switch not only in the miRNA expression patterns but also a pervasive shift in mRNA steady state levels of a number of genes to embryonic stage. Intriguingly, we found that loss of MBNL1 or gain of CELF1 activity, two major RNA binding proteins disrupted in DM1, are not driving the miRNA misregulation since their expression is indistinguishable between wild type, MBNL1 knock out and CELF1 over expressing mice. Moreover, comparable decrease in ten out of ten primary miRNA transcripts examined suggests loss of expression is not due to a processing defect. Instead, we discovered that adult-to-embryonic shift in expression of select micro- and messenger RNAs in DM1 heart occurs due to specific inactivation of a Mef2 transcriptional program. We are currently determining causal contributions of this Mef2-miRNA circuitry in the developmental reprogramming of gene expression in DM1 as well as its direct role in cardiac manifestations of this disease.

scholarly journals Increased Muscleblind levels by chloroquine treatment improve myotonic dystrophy type 1 phenotypes in in vitro and in vivo models

2019 ◽  
Vol 116 (50) ◽  
pp. 25203-25213 ◽  
Author(s):  
Ariadna Bargiela ◽  
Maria Sabater-Arcis ◽  
Jorge Espinosa-Espinosa ◽  
Miren Zulaica ◽  
Adolfo Lopez de Munain ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Trinucleotide Repeat ◽  
Rna Binding Proteins ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Muscle Area ◽  
In Vivo Models

Myotonic dystrophy type 1 (DM1) is a life-threatening and chronically debilitating neuromuscular disease caused by the expansion of a CTG trinucleotide repeat in the 3′ UTR of the DMPK gene. The mutant RNA forms insoluble structures capable of sequestering RNA binding proteins of the Muscleblind-like (MBNL) family, which ultimately leads to phenotypes. In this work, we demonstrate that treatment with the antiautophagic drug chloroquine was sufficient to up-regulate MBNL1 and 2 proteins in Drosophila and mouse (HSALR) models and patient-derived myoblasts. Extra Muscleblind was functional at the molecular level and improved splicing events regulated by MBNLs in all disease models. In vivo, chloroquine restored locomotion, rescued average cross-sectional muscle area, and extended median survival in DM1 flies. In HSALR mice, the drug restored muscular strength and histopathology signs and reduced the grade of myotonia. Taken together, these results offer a means to replenish critically low MBNL levels in DM1.

scholarly journals Automated High-Content Screening for Compounds That Disassemble the Perinucleolar Compartment

2009 ◽  
Vol 14 (9) ◽  
pp. 1045-1053 ◽  
Author(s):  
John T. Norton ◽  
Steven A. Titus ◽  
Dwayne Dexter ◽  
Christopher P. Austin ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Cellular Proliferation ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
High Content Screening ◽  
Screening Assay ◽  
Phenotypic Marker ◽  
Perinucleolar Compartment

All solid malignancies share characteristic traits, including unlimited cellular proliferation, evasion of immune regulation, and the propensity to metastasize. The authors have previously described that a subnuclear structure, the perinucleolar compartment (PNC), is associated with the metastatic phenotype in solid tumor cancer cells. The percentage of cancer cells that contain PNCs (PNC prevalence) is indicative of the malignancy of a tumor both in vitro and in vivo, and thus PNC prevalence is a marker that reflects metastatic capability in a population of tumor cells. Although the function of the PNC remains to be determined, the PNC is highly enriched with small RNAs and RNA binding proteins. The initial chemical biology studies using a set of anticancer drugs that disassemble PNCs revealed a direct association of the structure with DNA. Therefore, PNC prevalence reduction as a phenotypic marker can be used to identify compounds that target cellular processes required for PNC maintenance and hence used to elucidate the nature of the PNC function. Here the authors report the development of an automated high-content screening assay that is capable of detecting PNC prevalence in prostate cancer cells (PC-3M) stably expressing a green fluorescent protein (GFP)—fusion protein that localizes to the PNC. The assay was optimized using known PNC-reducing drugs and non-PNC-reducing cytotoxic drugs. After optimization, the fidelity of the assay was probed with a collection of 8284 compounds and was shown to be robust and capable of detecting known and novel PNC-reducing compounds, making it the first reported high-content phenotypic screen for small changes in nuclear structure. ( Journal of Biomolecular Screening 2009:1045-1053)

scholarly journals Ectopic expression ofDrosophilaELAV and human HuD inDrosophilawing disc cells reveals functional distinctions and similarities

2002 ◽  
Vol 115 (11) ◽  
pp. 2413-2421 ◽  
Author(s):  
Gakuta Toba ◽  
Jan Qui ◽  
Sandhya P. Koushika ◽  
Kalpana White
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Rna Binding Proteins ◽  
Mrna Level ◽  
Ectopic Expression ◽  
Wing Disc ◽  
Transcript Stability ◽  
Endogenous Expression ◽  
Disc Cells

Drosophila ELAV and human HuD are two neuronal RNA binding proteins that show remarkable sequence homology, yet differ in their respective documented roles in post-transcriptional regulation. ELAV regulates neural-specific alternative splicing of specific transcripts, and HuD stabilizes specific mRNAs that are otherwise unstable due to AU-rich elements(AREs) in their 3′ untranslated region (UTR). AREs are major determinants of transcript stability in mammalian cells. The role of each of these proteins was investigated and compared, by ectopically expressing them in Drosophila imaginal wing disc cells, which lack endogenous expression of either protein. The effect of the ectopic expression of ELAV and HuD was assessed on two sets of green fluorescent protein reporter transgenes,which were all driven with a broadly expressing promoter. Each set consisted of three reporter transgenes: (1) with an uninterrupted open reading frame(ORF); (2) with a constitutively spliced intron inserted into the ORF; and (3)with the intron nASI whose splicing is regulated in neurons by ELAV,inserted into the ORF. The two sets differed from each other only in their 3′UTR: Heat-shock-protein-70Ab (Hsp70Ab) trailer with ARE-like characteristics or Actin 5C (Act5C) trailer. Our results show that:(1) both ectopically expressed ELAV and HuD can enhance expression of transgenes with the Hsp70Ab 3′UTR, but not of transgenes with Act5C 3′UTR; (2) this enhancement is accompanied by an increase in mRNA level; (3) only ELAV can induce neural-specific splicing of nASI; and (4) although HuD is localized primarily to the cytoplasm,ELAV is localized to both the cytoplasm and the nucleus.

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