Functional VEGFA knockdown with artificial 3′-tailed mirtrons defined by 5′ splice site and branch point

Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons – 3′-tailed mirtrons – has hairpins precisely defined on the 5′ end by the 5′ splice site and 3′ end by the branch point. Here, we present design principles for artificial 3′-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy.     

Myotonic dystrophy: the role of the CUG triplet repeats in splicing of a novel DMPK exon and altered cytoplasmic DMPK mRNA isoform ratios

The mechanism by which (CTG)n expansion in the 3' UTR of the DMPK gene causes myotonic dystrophy (DM) is unknown. We identified four RNA splicing factors--hnRNP C, U2AF (U2 auxiliary factor), PTB (polypyrimidine tract binding protein), and PSF (PTB associated splicing factor)--that bind to two short regions 3' of the (CUG)n, and found a novel 3' DMPK exon resulting in an mRNA lacking the repeats. We propose that the (CUG)n is an essential cis acting element for this splicing event. In contrast to (CUG)n containing mRNAs, the novel isoform is not retained in the nucleus in DM cells, resulting in imbalances in relative levels of cytoplasmic DMPK mRNA isoforms and a new dominant effect of the mutation on DMPK.     

Myotonic dystrophy: RNA pathogenesis comes into focus

Myotonic dystrophy (DM)--the most common form of muscular dystrophy in adults, affecting 1/8000 individuals--is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system. Two genetic loci have been associated with the DM phenotype: DM1, on chromosome 19, and DM2, on chromosome 3. In 1992, the mutation responsible for DM1 was identified as a CTG expansion located in the 3' untranslated region of the dystrophia myotonica-protein kinase gene (DMPK). How this untranslated CTG expansion causes myotonic dystrophy type 1(DM1) has been controversial. The recent discovery that myotonic dystrophy type 2 (DM2) is caused by an untranslated CCTG expansion, along with other discoveries on DM1 pathogenesis, indicate that the clinical features common to both diseases are caused by a gain-of-function RNA mechanism in which the CUG and CCUG repeats alter cellular function, including alternative splicing of various genes. We discuss the pathogenic mechanisms that have been proposed for the myotonic dystrophies, the clinical and molecular features of DM1 and DM2, and the characterization of murine and cell-culture models that have been generated to better understand these diseases.     

Rationally designed small molecules targeting the RNA that causes myotonic dystrophy type 1 are potently bioactive

RNA is an important drug target, but it is difficult to design or discover small molecules that modulate RNA function. In the present study, we report that rationally designed, modularly assembled small molecules that bind the RNA that causes myotonic dystrophy type 1 (DM1) are potently bioactive in cell culture models. DM1 is caused when an expansion of r(CUG) repeats, or r(CUG)(exp), is present in the 3' untranslated region (UTR) of the dystrophia myotonica protein kinase (DMPK) mRNA. r(CUG)(exp) folds into a hairpin with regularly repeating 5'CUG/3'GUC motifs and sequesters muscleblind-like 1 protein (MBNL1). A variety of defects are associated with DM1, including (i) formation of nuclear foci, (ii) decreased translation of DMPK mRNA due to its nuclear retention, and (iii) pre-mRNA splicing defects due to inactivation of MBNL1, which controls the alternative splicing of various pre-mRNAs. Previously, modularly assembled ligands targeting r(CUG)(exp) were designed using information in an RNA motif-ligand database. These studies showed that a bis-benzimidazole (H) binds the 5'CUG/3'GUC motif in r(CUG)(exp.) Therefore, we designed multivalent ligands to bind simultaneously multiple copies of this motif in r(CUG)(exp). Herein, we report that the designed compounds improve DM1-associated defects including improvement of translational and pre-mRNA splicing defects and the disruption of nuclear foci. These studies may establish a foundation to exploit other RNA targets in genomic sequence.     

Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.     

Myotonic dystrophy type 1 (DM1): A triplet repeat expansion disorder

Myotonic dystrophy is a progressive multisystem genetic disorder affecting about 1 in 8000 people worldwide. The unstable repeat expansions of (CTG)n or (CCTG)n in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy: (i) myotonic dystrophy type 1 (DM1) and (ii) myotonic dystrophy type 2 (DM2) respectively. There is currently no cure but supportive management helps equally to reduce the morbidity and mortality and patients need close follow up to pay attention to their clinical problems. This review will focus on the clinical features, molecular view and genetics, diagnosis and management of DM1.     

Myotonic dystrophy protein kinase (DMPK) and its role in the pathogenesis of myotonic dystrophy 1

Myotonic dystrophy 1 (DM1) is an autosomal, dominant inherited, neuromuscular disorder. The DM1 mutation consists in the expansion of an unstable CTG-repeat in the 3'-untranslated region of a gene encoding DMPK (myotonic dystrophy protein kinase). Clinical expression of DM1 is variable, presenting a progressive muscular dystrophy that affects distal muscles more than proximal and is associated with the inability to relax muscles appropriately (myotonia), cataracts, cardiac arrhythmia, testicular atrophy and insulin resistance. DMPK is a Ser/Thr protein kinase homologous to the p21-activated kinases MRCK and ROCK/rho-kinase/ROK. The most abundant isoform of DMPK is an 80 kDa protein mainly expressed in smooth, skeletal and cardiac muscles. Decreased DMPK protein levels may contribute to the pathology of DM1, as revealed by gene target studies. Here we review current understanding of the structural, functional and pathophysiological characteristics of DMPK.     

DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1

Myotonic dystrophy type 1 (DM1) is caused by CUG triplet expansions in the 3′ UTR of dystrophia myotonica protein kinase (DMPK) messenger ribonucleic acid (mRNA). The etiology of this multi-systemic disease involves pre-mRNA splicing defects elicited by the ability of the CUG-expanded mRNA to ‘sponge’ splicing factors of the muscleblind family. Although nuclear aggregation of CUG-containing mRNPs in distinct foci is a hallmark of DM1, the mechanisms of their homeostasis have not been completely elucidated. Here we show that a DEAD-box helicase, DDX6, interacts with CUG triplet-repeat mRNA in primary fibroblasts from DM1 patients and with CUG–RNA in vitro. DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci. Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus. While the level of CUG-expanded mRNA is unaffected by increased DDX6 expression, the mRNA re-localizes to the cytoplasm and its interaction partner MBNL1 becomes dispersed and also partially re-localized to the cytoplasm. Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.     

Myotonic dystrophy protein kinase is critical for nuclear envelope integrity

Myotonic dystrophy 1 (DM1) is a multisystemic disease caused by a triplet nucleotide repeat expansion in the 3' untranslated region of the gene coding for myotonic dystrophy protein kinase (DMPK). DMPK is a nuclear envelope (NE) protein that promotes myogenic gene expression in skeletal myoblasts. Muscular dystrophy research has revealed the NE to be a key determinant of nuclear structure, gene regulation, and muscle function. To investigate the role of DMPK in NE stability, we analyzed DMPK expression in epithelial and myoblast cells. We found that DMPK localizes to the NE and coimmunoprecipitates with Lamin-A/C. Overexpression of DMPK in HeLa cells or C2C12 myoblasts disrupts Lamin-A/C and Lamin-B1 localization and causes nuclear fragmentation. Depletion of DMPK also disrupts NE lamina, showing that DMPK is required for NE stability. Our data demonstrate for the first time that DMPK is a critical component of the NE. These novel findings suggest that reduced DMPK may contribute to NE instability, a common mechanism of skeletal muscle wasting in muscular dystrophies.