Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5

Abstract

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are neuromuscular diseases, caused by accumulation of CUG and CCUG RNAs in toxic aggregates. Here we report that the increased stability of the mutant RNAs in both types of DM is caused by deficiency of RNA helicase p68. We have identified p68 by studying CCUG-binding proteins associated with degradation of the mutant CCUG repeats. Protein levels of p68 are reduced in DM1 and DM2 biopsied skeletal muscle. Delivery of p68 in DM1/2 cells causes degradation of the mutant RNAs, whereas delivery of p68 in skeletal muscle of DM1 mouse model reduces skeletal muscle myopathy and atrophy. Our study shows that correction of p68 may reduce toxicity of the mutant RNAs in DM1 and in DM2.myotonic dystrophy | RNA foci | p68 RNA helicase | CUG repeats M yotonic dystrophy type 1 (DM1) is a neuromuscular disease characterized by myotonia, distal muscle weakness, heart conduction defects, and, in the congenital form, a delay in myogenesis and severe cognitive abnormalities (1). DM1 is caused by expanded CTG repeats within the 3′ untranslated region of the DMPK gene (2). Myotonic dystrophy type 2 (DM2) is a late-onset disease that is caused by expanded CCTG repeats in intron 1 of the ZNF9/CNBP gene (3). Development of therapeutic approaches for DM1 or DM2 is an urgent need. Numerous data suggest that DM1 and DM2 are caused by RNA gain-offunction mechanisms (4-6). Initial studies showed that mutant RNAs mainly affect two RNA-binding proteins, CUG-binding protein 1 (CUGBP1) and muscleblind-like protein 1 (MBNL1) (7-9). CUG repeats elevate protein levels of CUGBP1 by increasing its stability (5). In addition, CUG repeats change signal transduction pathways, such as the glycogen synthase kinase 3β (GSK3β)-cyclin D3 pathway, regulating CUGBP1 activity (5, 10). CUG and CCUG repeats form double-stranded hairpin structures and sequester MBNL1 (9,11,12). Several other RNA-binding proteins, such as Staufen1 and two members of the DEAD-box RNA helicases family, DDX5/p68 and DDX6, are also involved in DM1 (13-15).We showed that the mutant CUG and CCUG RNAs are very stable (16), suggesting that the activity of RNA-binding proteins regulating RNA decay is reduced in DM1 and in DM2. In this study, we tested this hypothesis by isolation and analysis of several CCUG-binding proteins. We found that the levels of one of these proteins, p68, are reduced in DM1 and DM2 biopsied muscle and that correction of p68 leads to degradation of the mutant CUG and CCUG RNAs, disintegration of RNA foci, and reduction of DM muscle pathology. Results and DiscussionIdentification of p68 Helicase as CUG/CCUG-Binding Protein, Which is Reduced During Degradation of CCUG Repeats. Given the critical role of RNA CUG and CCUG repeats in DM1/2, we performed a careful analysis of CCUG-binding proteins with altered activities upon accumulation and degradation of the mutant CCUG repeats in tetracycline (tet)-regulated CHO cell model of DM2. In this model, CCUG 100 repeats mainly increased activities of two RNA-binding proteins ...

The RNA binding KH domain of Spoonbill depletes pathogenic non-coding spinocerebellar ataxia 8 transcripts and suppresses neurodegeneration in Drosophila

“…Biopsy samples from DM1 and DM2 skeletal muscles showed the reduced level of p68. Ectopic p68 reduces skeletal muscle myopathy and atrophy by lowering the pathogenic CUG or CCUG RNA(49). First, Spoon KH domain might be facilitating decay of the pathogenic SCA8 transcripts.…”

RNA toxicity and foci formation in microsatellite expansion diseases

“…For instance, two DM1 RNA toxic foci enhancers ( str-67 and ocrl-1 ) have been identified in Caenorhabditis elegans [93]. Nonsense-mediated decay, Staufen and DEAD-box helicase 5 (DDX5) all have an impact on toxic RNA transport and degradation [94–96]. In addition, a large ribonucleoprotein complex may transiently regulate the sense foci (Fig.…”

Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1

“…Degradation of the mutant DMPK mRNA using antisense oligonucleotides or by increase of RNA helicase p68 corrects muscle pathology in HSA LR mice (10, 12). Normalization of the activities of RNA‐binding proteins, misregulated by CUG repeats (CUGBP1 and MBNL1), was also shown to be beneficial for the reduction of DM1 muscle pathology in HSA LR mice (12).…”

“…Two of the most-studied proteins, associated with the disruption of RNA processing in DM1, are muscleblind 1 (MBNL1) and CUGBP1 [(also known as CUGBP1 elav-like factor (CELF1)] (3)(4)(5)(6)(7). Other RNA-binding proteins are also affected in DM1, including Staufen1 and RNA helicases DDX5 and DDX6 (8)(9)(10)(11).…”

“…ABBREVIATIONS: BIO, 6-bromoindirubin-39-oxime; CDK4, cyclin Ddependent kinase 4; CDM1, congenital myotonic dystrophy type 1; Col, collagen; CUGBP1/CELF1, CUG-binding protein/CUGBP1 elav-like factor 1; DCX, doublecortin; DDX, DEAD-box; DM1, myotonic dystrophy type 1; DMPK, dystrophia myotonica protein kinase; ECM, extracellular matrix; eIF2, eukaryotic initiation factor 2; ES, embryonic stem cell; GSK3b, glycogen synthase kinase 3b; het, heterozygous; hom, homozygous; HSA LR , human skeletal actin mRNA, containing long repeats; IF, immunofluorescence; IP, immunoprecipitation; KO, knockout; LEF1, lymphoid enhancer binding factor 1; MBNL1, muscleblind 1 protein; MSC, myogenic satellite cell; p-, phosphorylated; Pax7, paired box 7 factor; Ppp1r14d, protein phosphatase 1 regulatory inhibitor subunit 14D; RBM45, RNA-binding motif 45 protein; RRM, RNA recognition motif; RUNX2, runt-related transcription factor 2; TCF7, T cell specific transcription factor 7; TDZD-8, 4-benzyl-2-methyl-1,2,4thiadiazolidine-3,5-dione; unp, unphosphorylated; WT, wild type Degradation of the mutant DMPK mRNA using antisense oligonucleotides or by increase of RNA helicase p68 corrects muscle pathology in HSA LR mice (10,12). Normalization of the activities of RNA-binding proteins, misregulated by CUG repeats (CUGBP1 and MBNL1), was also shown to be beneficial for the reduction of DM1 muscle pathology in HSA LR mice (12).…”