The RNA-binding Protein CUGBP1 Regulates Stability of Tumor Necrosis Factor mRNA in Muscle Cells

Abstract

Type I myotonic dystrophy (DM1) is caused by a triplet repeat expansion in the 3-untranslated region (UTR) of the dystrophia myotonia protein kinase (DMPK) gene. Pathogenesis is closely linked with production of a toxic RNA from the mutant allele, which interferes with function of several RNA-binding proteins, including CUGBP1. Here we show that expression of a mutant DMPK 3-UTR containing 960 CUG repeats is sufficient to increase expression and stability of an mRNA encoding the potent proinflammatory cytokine, tumor necrosis factor (TNF). CUGBP1 specifically recognizes sequences within the TNF 3-UTR that are dissimilar from its canonical UG-rich binding site. Depletion of CUGBP1 from mouse myoblasts results in increased abundance of TNF mRNA through stabilization of the transcript. Moreover, activation of the protein kinase C pathway by treatment with phorbol ester, which has been shown previously to result in CUGBP1 phosphorylation, also causes TNF mRNA stabilization. Our results suggest that the elevated serum TNF seen in DM1 patients may be derived from muscle where it is induced by expression of toxic DMPK RNA. Importantly, overexpression of this potent cytokine could contribute to the muscle wasting and insulin resistance that are characteristic of this debilitating disease. Myotonic dystrophy type I (DM1)3 is a debilitating autosomal dominant disorder caused by a triplet CTG repeat expansion in the 3Ј-untranslated region (UTR) of the dystrophin myotonia protein kinase (DMPK) gene (1-3). The disease is characterized by myotonia, progressive muscle wasting, insulin resistance, and cardiac conduction defects (4). Although some of the symptoms of DM1 may be attributed to reduced levels of the DMPK protein, much of the disease pathogenesis is recapitulated by expression of the mutant DMPK 3Ј-UTR (5, 6), or even by elevated expression of the wild type DMPK 3Ј-UTR (7), in otherwise normal mice. This is, at least in part, because transcripts containing the expanded CUG repeat accumulate in nuclear foci where they sequester cellular proteins (8).The function of two RNA-binding proteins, CUGBP1 (CUGbinding protein 1) and MBNL (Muscleblind), is profoundly affected in DM1 patient cells. MBNL associates with the repeatcontaining mRNA and is confined to nuclear foci (8), whereas CUGBP1 is overexpressed and hyperphosphorylated in the nucleus (9, 10). There is also evidence that CUGBP1 may be depleted from the cytoplasm (10). Interestingly, CUGBP1 and MBNL are both splicing regulators that play antagonistic roles in splice site selection. Consistent with this, several clinically relevant mRNAs, including the muscle-specific chloride channel (11), the insulin receptor (12), and cardiac troponin T (13), exhibit aberrant splice patterns in DM1 patient cells.Several studies have indicated that aberrant function of CUGBP1 is a key factor in development of DM1 (5, 6, 14), and it is therefore important to consider other cellular functions of this protein that may be affected. It has been known for some time that CUGBP1 regulate...

Regulation of CUG-binding Protein 1 (CUGBP1) Binding to Target Transcripts upon T Cell Activation

“…In a myotonic dystrophy model, CUGBP1 was hyperphosphorylated by PKC␣ and ␤II, causing increased protein half-life and altered splicing patterns (19,30). Expression of a (CUG) 960 -expanded myotonic dystrophy protein kinase gene caused CUGBP1 phosphorylation and inhibition of CUGBP1-mediated decay of the TNF-␣ transcript (21). It is thought that CUGBP1 binds to a CG-rich motif in the (24).…”

“…In a mouse model of myotonic dystrophy, it has been shown that CUGBP1 is phosphorylated by protein kinase C (PKC), resulting in altered cellular distribution and stability of the CUGBP1 protein, correlating with altered splicing patterns (19,20). The CUGBP1 target transcript TNF-␣ was stabilized upon chemical activation of the PKC pathway (21). In addition, the RNA binding specificity of CUGBP1 has been shown to be altered via phosphorylation by cyclin D3-Cdk4/6 (22).…”

Myotonic Dystrophy Type 1: Focus on the RNA Pathology and Therapy

“…Moreover, activation of the protein kinase C (PKC) pathway also stabilized the TNF transcript. These results suggest that the elevated serum TNF seen in DM1 patients may be derived from muscle where it is induced by expression of toxic DMPK RNA (Zhang et al, 2008). In a more recent study, Koshelev et al…”

“…Experiments performed in C2C12 mouse cell line showed that expression of a mutant DMPK 3'-UTR containing 960 CUG repeats is sufficient to increase expression and stability of an mRNA encoding the potent proinflammatory cytokine, tumor necrosis factor (TNF), which was found elevated in DM1 patients serum (Mammarella et al, 2002;Zhang et al, 2008). Moreover, activation of the protein kinase C (PKC) pathway also stabilized the TNF transcript.…”

CELF1, a Multifunctional Regulator of Posttranscriptional Networks

“…In the DM1 disease model, there is aberrant activation of the protein kinase C pathway as a result of the CTG expansion, and this results in CELF1 phosphorylation. Mouse myoblasts (C2C12 cells) made to express CTG expanded RNA were shown to experience stabilization of tumor necrosis factor alpha (TNF-alpha) mRNA [143]. This result suggested that the overexpression of TNF-alpha observed in DM1 could be coming from muscle, and this TNFalpha overexpression may contribute to the muscle wasting and insulin resistance that are characteristic of this disease [143].…”