Altered phosphorylation and intracellular distribution of a (CUG) _n triplet repeat RNA-binding protein in patients with myotonic dystrophy and in myotonin protein kinase knockout mice

Abstract

Myotonic dystrophy (DM) is associated with expansion of CTG repeats in the 3-untranslated region of the myotonin protein kinase (DMPK) gene. The molecular mechanism whereby expansion of the (CUG) n repeats in the 3-untranslated region of DMPK gene induces DM is unknown. We previously isolated a protein with specific binding to CUG repeat sequences (CUG-BP͞hNab50) that possibly plays a role in mRNA processing and͞or transport. Here we present evidence that the phosphorylation status and intracellular distribution of the RNA CUG-binding protein, identical to hNab50 protein (CUG-BP͞hNab50), are altered in homozygous DM patient and that CUG-BP͞hNab50 is a substrate for DMPK both in vivo and in vitro. Data from two biological systems with reduced levels of DMPK, homozygous DM patient and DMPK knockout mice, show that DMPK regulates both phosphorylation and intracellular localization of the CUG-BP͞hNab50 protein. Decreased levels of DMPK observed in DM patients and DMPK knockout mice led to the elevation of the hypophosphorylated form of CUG-BP͞ hNab50. Nuclear concentration of the hypophosphorylated CUG-BP͞hNab50 isoform is increased in DMPK knockout mice and in homozygous DM patient. DMPK also interacts with and phosphorylates CUG-BP͞hNab50 protein in vitro. DMPK-mediated phosphorylation of CUG-BP͞hNab50 results in dramatic reduction of the CUG-BP2, hypophosphorylated isoform, accumulation of which was observed in the nuclei of DMPK knockout mice. These data suggest a feedback mechanism whereby decreased levels of DMPK could alter phosphorylation status of CUG-BP͞hNab50, thus facilitating nuclear localization of CUG-BP͞hNab50. Our results suggest that DM pathophysiology could be, in part, a result of sequestration of CUG-BP͞hNab50 and, in part, of lowered DMPK levels, which, in turn, affect processing and transport of specific subclass of mRNAs.Myotonic dystrophy (DM), the most common form of adult muscular dystrophy, although primarily a muscle disorder, also affects other organs including the heart, eye, testes, lungs, and brain (1). The primary genetic defect in DM is a (CTG) n trinucleotide repeat expansion in the 3Ј-untranslated region of the myotonin-protein kinase (DMPK) gene (2-7). The length of this expansion correlates with the severity of the clinical symptoms (8-10). The mechanism by which the CTG repeat expansion induces the DM phenotype remains puzzling, particularly because the CTG repeat is located in the 3Ј-untranslated region. In homozygous knockout mice, where DMPK is absent, only muscle weakness and myopathy are observed (11,12). Cardiomyopathy develops in mice overexpressing the DMPK gene (12), but disease symptoms in other organs, including skeletal muscle myotonia are not observed. These data show that alteration of DMPK expression is only a part of DM mechanism suggesting that other genes are involved.In pursuit of alternative mechanisms for the pathogenesis of DM, we identified novel proteins that bind specifically to triplet repeats (13) in DNA and others that bind to triplet repeat...

c-Jun ARE Targets mRNA Deadenylation by an EDEN-BP (Embryo Deadenylation Element-binding Protein)-dependent Pathway

“…However, CUG-BP has been detected both in the nucleus and the cytoplasm (32). Moreover, most ARE-binding factors are nucleus-cytoplasm shuttling proteins, with different functions in these two compartments (reviewed in Ref.…”

Viral vector producing antisense RNA restores myotonic dystrophy myoblast functions

“…[11][12][13] This deregulation of CUGBP1 results in delay of skeletal muscle differentiation and insulin resistance in DM1 disease. Since the expression of antisense RNA restored efficiency of muscle differentiation and glucose uptake (Figures 4 and 5, and Table 1), we next examined whether the antisense RNA brings CUGBP1 expression to the normal levels.…”

“…[2][3][4] At the molecular levels, the DM1 phenotype is most likely caused by a complex molecular pathogenesis, including deficiency of myotonic dystrophy myotonin kinase (DMPK) protein, [5][6][7] haplo-insufficiency of a neighboring homeobox gene (particular the DM locus-associated homeodomain protein (DMAHP/Six 5 gene) 8 and the WD-repeat gene (DMWD) 9 and a trans-dominant misregulation of RNA homeostasis. [10][11][12][13][14][15][16] Recent experiments from transgenic mice, expressing an untranslated expanded CUG repeat under the control of the human skeletal actin promoter, showed that expanded CUG repeats are sufficient to generate DM1 muscle phenotype. 17 These data suggest that misregulation of RNA homeostasis may play a major role in DM1 muscle pathogenesis.…”

Overexpression of CUG Triplet Repeat-binding Protein, CUGBP1, in Mice Inhibits Myogenesis

“…The mechanisms by which the transgenic His-CUGBP1 protein induces protein levels of endogenous CUGBP1 are unknown. Our data also showed that CUGBP1 immunoreactive proteins migrate as several bands, perhaps because of different levels in phosphorylation (24). To examine whether these immunoreactive bands are isoforms of His-CUGBP1, we immunoprecipitated His-CUGBP1 with polyclonal antibodies to His and performed a two-dimensional gel electrophoresis followed by Western blotting with monoclonal antibodies to CUGBP1.…”