scholarly journals Cpy(a/t)(q/w)e D-Hexapeptides Bind CUG Repeats and Rescue Phenotypes of Myotonic Dystrophy Myoblasts and A Drosophila Model of the Disease

2020 ◽  
Author(s):  
Ariadna Bargiela ◽  
Ruben Artero ◽  
Roger Estrada-Tejedor ◽  
Anna Rapisarda ◽  
Enrique Garcia-España ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ctg Repeats ◽  
Rna Hairpins ◽  
Drosophila Model ◽  
Toxic Rna ◽  
Rna Complexes ◽  
Cug Repeats

Abstract In Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic double-stranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects. In this paper, we characterize four D-amino acid hexapeptides identified together with abp1, a peptide previously shown to stabilize CUG RNA in its single-stranded conformation. With the generalized sequence cpy(a/t)(q/w)e, these related peptides improved three MBNL-regulated exon inclusions in DM1-derived cells. Subsequent experiments showed that these compounds generally increased the relative expression of MBNL1 and its nuclear-cytoplasmic distribution, reduced hyperactivated autophagy, and increased the percentage of differentiated (Desmin-positive) cells in vitro. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests. Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different constant affinities, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp1. Finally, molecular modeling suggests a detailed view of the interactions of peptide-CUG RNA complexes useful in the chemical optimization of compounds.

scholarly journals Defined d-hexapeptides bind CUG repeats and rescue phenotypes of myotonic dystrophy myotubes in a Drosophila model of the disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna Rapisarda ◽  
Ariadna Bargiela ◽  
Beatriz Llamusi ◽  
Isabel Pont ◽  
Roger Estrada-Tejedor ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Ctg Repeats ◽  
Rna Hairpins ◽  
Drosophila Model ◽  
Toxic Rna ◽  
Rna Complexes ◽  
Cug Repeats

AbstractIn Myotonic Dystrophy type 1 (DM1), a non-coding CTG repeats rare expansion disease; toxic double-stranded RNA hairpins sequester the RNA-binding proteins Muscleblind-like 1 and 2 (MBNL1 and 2) and trigger other DM1-related pathogenesis pathway defects. In this paper, we characterize four d-amino acid hexapeptides identified together with abp1, a peptide previously shown to stabilize CUG RNA in its single-stranded conformation. With the generalized sequence cpy(a/t)(q/w)e, these related peptides improved three MBNL-regulated exon inclusions in DM1-derived cells. Subsequent experiments showed that these compounds generally increased the relative expression of MBNL1 and its nuclear-cytoplasmic distribution, reduced hyperactivated autophagy, and increased the percentage of differentiated (Desmin-positive) cells in vitro. All peptides rescued atrophy of indirect flight muscles in a Drosophila model of the disease, and partially rescued muscle function according to climbing and flight tests. Investigation of their mechanism of action supports that all four compounds can bind to CUG repeats with slightly different association constant, but binding did not strongly influence the secondary structure of the toxic RNA in contrast to abp1. Finally, molecular modeling suggests a detailed view of the interactions of peptide-CUG RNA complexes useful in the chemical optimization of compounds.

scholarly journals Correction of RNA-Binding Protein CUGBP1 and GSK3β Signaling as Therapeutic Approach for Congenital and Adult Myotonic Dystrophy Type 1

2019 ◽  
Vol 21 (1) ◽  
pp. 94 ◽  
Author(s):  
Lubov Timchenko
Keyword(s):  
Myotonic Dystrophy ◽  
Complex Disease ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Myotonic Dystrophy Type 1 ◽  
Myotonic Dystrophy Type ◽  
Ctg Repeats ◽  
Toxic Rna

Myotonic dystrophy type 1 (DM1) is a complex genetic disease affecting many tissues. DM1 is caused by an expansion of CTG repeats in the 3′-UTR of the DMPK gene. The mechanistic studies of DM1 suggested that DMPK mRNA, containing expanded CUG repeats, is a major therapeutic target in DM1. Therefore, the removal of the toxic RNA became a primary focus of the therapeutic development in DM1 during the last decade. However, a cure for this devastating disease has not been found. Whereas the degradation of toxic RNA remains a preferential approach for the reduction of DM1 pathology, other approaches targeting early toxic events downstream of the mutant RNA could be also considered. In this review, we discuss the beneficial role of the restoring of the RNA-binding protein, CUGBP1/CELF1, in the correction of DM1 pathology. It has been recently found that the normalization of CUGBP1 activity with the inhibitors of GSK3 has a positive effect on the reduction of skeletal muscle and CNS pathologies in DM1 mouse models. Surprisingly, the inhibitor of GSK3, tideglusib also reduced the toxic CUG-containing RNA. Thus, the development of the therapeutics, based on the correction of the GSK3β-CUGBP1 pathway, is a promising option for this complex disease.

scholarly journals Staufen1 impairs stress granule formation in skeletal muscle cells from myotonic dystrophy type 1 patients

2016 ◽  
Vol 27 (11) ◽  
pp. 1728-1739 ◽  
Author(s):  
Aymeric Ravel-Chapuis ◽  
Amanda Klein Gunnewiek ◽  
Guy Bélanger ◽  
Tara E. Crawford Parks ◽  
Jocelyn Côté ◽  
...  
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Muscle Cells ◽  
Cellular Stress Response ◽  
Granule Formation ◽  
A Cell ◽  
Cell Type Specific ◽  
Cug Repeats

Myotonic dystrophy (DM1) is caused by an expansion of CUG repeats (CUGexp) in the DMPK mRNA 3′UTR. CUGexp-containing mRNAs become toxic to cells by misregulating RNA-binding proteins. Here we investigated the consequence of this RNA toxicity on the cellular stress response. We report that cell stress efficiently triggers formation of stress granules (SGs) in proliferating, quiescent, and differentiated muscle cells, as shown by the appearance of distinct cytoplasmic TIA-1– and DDX3-containing foci. We show that Staufen1 is also dynamically recruited into these granules. Moreover, we discovered that DM1 myoblasts fail to properly form SGs in response to arsenite. This blockage was not observed in DM1 fibroblasts, demonstrating a cell type–specific defect. DM1 myoblasts display increased expression and sequestration of toxic CUGexp mRNAs compared with fibroblasts. Of importance, down-regulation of Staufen1 in DM1 myoblasts rescues SG formation. Together our data show that Staufen1 participates in the inhibition of SG formation in DM1 myoblasts. These results reveal that DM1 muscle cells fail to properly respond to stress, thereby likely contributing to the complex pathogenesis of DM1.

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 CELL NUCLEAR ALTERATIONS IN MYOTONIC DYSTROPHY

2014 ◽  
pp. 25-40
Author(s):  
Manuela Malatesta ◽  
Marzia Giagnacovo
Keyword(s):  
Myotonic Dystrophy ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Light And Electron Microscopy ◽  
In Vitro Study ◽  
Cardiac Conduction ◽  
Cellular Mechanisms ◽  
Transcript Processing ◽  
Nuclear Alterations

In the cell nucleus, genes are transcribed, and the primary transcripts undergo molecular processing which generates mature RNAs to be exported to the cytoplasm. The events leading to the formation of mature RNAs are chronologically and spatially ordered, and they mostly occur on distinct ribonucleoprotein (RNP)-containing structures. Defects in the RNA maturation pathways have been related to diseases leading to muscle dystrophy: in myotonic dystrophy type 1 (DM1) and type 2 (DM2), the characteristic multisystemic features (e.g., myotonia, muscular dystrophy, dilated cardiomyopathy, cardiac conduction defects, cataracts, insulin-resistance, and disease-specific serological abnormalities) are caused by the expansion of two distinct nucleotide sequences: (CTG)n in the 3’ untranslated region of the DMPK gene on chromosome 19q13 in DM1, and (CCTG)n in the first intron of the ZNF9 gene on chromosome 3q21 in DM2. Combining biomolecular and cytochemical techniques, it has been demonstrated that the basic mechanisms of both DMs reside in the nuclear sequestration of the expanded RNAs: CUG- and CCUG-containing transcripts accumulate in intranuclear foci in DM1 and DM2 cells respectively, and alter the regulation and intranuclear localization of the RNA-binding proteins CUGBP1 and MBLN, which are necessary for the physiological processing of pre-mRNA. Using immunocytochemical techniques at light and electron microscopy, we have demonstrated that MBNL1-containing foci in DM2 cells also sequester snRNPs and hnRNPs, splicing factors involved in the early phases of transcript processing; this strengthens the hypothesis that the multifactorial phenotype of dystrophic patients could be due to a general alteration of the pre-mRNA post-transcriptional pathway. Interestingly, we also demonstrated that, in skeletal muscles of DM1 and DM2 patients, splicing and cleavage factors accumulate in myonuclei, suggesting an impairment of pre-mRNA processing reminiscent of the nuclear alterations typical of sarcopenia (i.e. the loss of muscle mass and function physiologically occurring during ageing). Moreover, in an in vitro study, we observed that satellite-cell-derived DM2 myoblasts show cell senescence alterations and impairment of the pre-mRNA maturation pathways earlier than the myoblasts from healthy patient. These results suggest possible common cellular mechanisms responsible for skeletal muscle wasting in different pathologies.

scholarly journals C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons

2021 ◽  
Author(s):  
Eun Seon Kim ◽  
Chang Geon Chung ◽  
Jeong Hyang Park ◽  
Byung Su Ko ◽  
Sung Soon Park ◽  
...  
Keyword(s):  
Retinal Degeneration ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Heterozygous Mutation ◽  
Pathological Feature ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Drosophila Model ◽  
Post Transcriptional Regulation

Abstract RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP, Staufen, may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS—but not with mutated endogenous NLS—potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/−), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/− is protective. Stau+/− also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

scholarly journals Identification of mRNAs Associated with αCP2-Containing RNP Complexes

2003 ◽  
Vol 23 (19) ◽  
pp. 7055-7067 ◽  
Author(s):  
Shelly A. Waggoner ◽  
Stephen A. Liebhaber
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
Cell Function ◽  
Rna Binding Proteins ◽  
Translation Control ◽  
Viral Mrnas ◽  
Posttranscriptional Gene Regulation ◽  
Direct Binding

ABSTRACT Posttranscriptional controls in higher eukaryotes are central to cell differentiation and developmental programs. These controls reflect sequence-specific interactions of mRNAs with one or more RNA binding proteins. The α-globin poly(C) binding proteins (αCPs) comprise a highly abundant subset of K homology (KH) domain RNA binding proteins and have a characteristic preference for binding single-stranded C-rich motifs. αCPs have been implicated in translation control and stabilization of multiple cellular and viral mRNAs. To explore the full contribution of αCPs to cell function, we have identified a set of mRNAs that associate in vivo with the major αCP2 isoforms. One hundred sixty mRNA species were consistently identified in three independent analyses of αCP2-RNP complexes immunopurified from a human hematopoietic cell line (K562). These mRNAs could be grouped into subsets encoding cytoskeletal components, transcription factors, proto-oncogenes, and cell signaling factors. Two mRNAs were linked to ceroid lipofuscinosis, indicating a potential role for αCP2 in this infantile neurodegenerative disease. Surprisingly, αCP2 mRNA itself was represented in αCP2-RNP complexes, suggesting autoregulatory control of αCP2 expression. In vitro analyses of representative target mRNAs confirmed direct binding of αCP2 within their 3′ untranslated regions. These data expand the list of mRNAs that associate with αCP2 in vivo and establish a foundation for modeling its role in coordinating pathways of posttranscriptional gene regulation.

scholarly journals Function of Pumilio Genes in Human Embryonic Stem Cells and Their Effect in Stemness and Cardiomyogenesis

2019 ◽  
Author(s):  
Isabelle Leticia Zaboroski Silva ◽  
Anny Waloski Robert ◽  
Guillermo Cabrera Cabo ◽  
Lucia Spangenberg ◽  
Marco Augusto Stimamiglio ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Mrna Levels ◽  
Human Escs ◽  
Mrna Targets ◽  
Cardiomyogenic Differentiation

AbstractPosttranscriptional regulation plays a fundamental role in the biology of embryonic stem cells (ESCs). Many studies have demonstrated that multiple mRNAs are coregulated by one or more RNA binding proteins (RBPs) that orchestrate the expression of these molecules. A family of RBPs, known as PUF (Pumilio-FBF), is highly conserved among species and has been associated with the undifferentiated and differentiated states of different cell lines. In humans, two homologs of the PUF family have been found: Pumilio 1 (PUM1) and Pumilio 2 (PUM2). To understand the role of these proteins in human ESCs (hESCs), we first demonstrated the influence of the silencing of PUM1 and PUM2 on pluripotency genes. OCT4 and NANOG mRNA levels decreased significantly with the knockdown of Pumilio, suggesting that PUMILIO proteins play a role in the maintenance of pluripotency in hESCs. Furthermore, we observed that the hESCs silenced for PUM1 and 2 exhibited an improvement in efficiency of in vitro cardiomyogenic differentiation. Using in silico analysis, we identified mRNA targets of PUM1 and PUM2 expressed during cardiomyogenesis. With the reduction of PUM1 and 2, these target mRNAs would be active and could be involved in the progression of cardiomyogenesis.

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