The purpose of this study was to investigate the role of the mutant CUGn RNA in the induction of stress in type 1 myotonic dystrophy (DM1) cells and in the stress-mediated inhibition of protein translation in DM1. To achieve our goals, we performed HPLC-based purification of stress granules (SGs), immunoanalysis of SGs with stress markers TIA-1, CUGBP1, and ph-eIF2, site-specific mutagenesis, and examinations of RNA-protein and protein-protein interactions in myoblasts from control and DM1 patients. The cause-and-effect relationships were addressed in stable cells expressing mutant CUG repeats. We found that the mutant CUGn RNA induces formation of SGs through the increase of the double-stranded RNA-dependent protein kinase (PKR) and following inactivation of eIF2α, one of the substrates of PKR. We show that SGs trap mRNA coding for the DNA repair and remodeling factor MRG15 (MORF4L1), translation of which is regulated by CUGBP1. As the result of the trapping, the levels of MRG15 are reduced in DM1 cells and in CUG-expressing cells. These data show that CUG repeats cause stress in DM1 through the PKR-ph-eIF2α pathway inhibiting translation of certain mRNAs, such as MRG15 mRNA. The repression of protein translation by stress might contribute to the progressive muscle loss in DM1.

Differentiation of myocytes is impaired in patients with myotonic dystrophy type 1, DM1. CUG repeat binding protein, CUGBP1, is a key regulator of translation of proteins that are involved in muscle development and differentiation. In this paper, we present evidence that RNA-binding activity of CUGBP1 and its interactions with initiation translation complex eIF2 are differentially regulated during myogenesis by specific phosphorylation and that this regulation is altered in DM1. In normal myoblasts, Akt kinase phosphorylates CUGBP1 at Ser28 and increases interactions of CUGBP1 with cyclin D1 mRNA. During differentiation, CUGBP1 is phosphorylated by cyclinD3-cdk4/6 at Ser302, which increases CUGBP1 binding with p21 and C/EBPbeta mRNAs. While cyclin D3 and cdk4 are elevated in normal myotubes; DM1 differentiating cells do not increase these proteins. In normal myotubes, CUGBP1 interacts with cyclin D3/cdk4/6 and eIF2; however, interactions of CUGBP1 with eIF2 are reduced in DM1 differentiating cells and correlate with impaired muscle differentiation in DM1. Ectopic expression of cyclin D3 in DM1 cells increases the CUGBP1-eIF2 complex, corrects expression of differentiation markers, myogenin and desmin, and enhances fusion of DM1 myoblasts. Thus, normalization of cyclin D3 might be a therapeutic approach to correct differentiation of skeletal muscle in DM1 patients.

Myotonic Dystrophy 2 (DM2) is an autosomal dominant, multisystem disease, which primarily affects skeletal muscle. DM2 is caused by CCTGn expansion in the intron 1 of the ZNF9 gene. Expression of the mutant CCUGn RNA changes RNA processing in patients with DM2; however, the role of ZNF9 protein in DM2 pathology has been not elucidated. ZNF9 has been shown to regulate cap-dependent and cap-independent translation. We have examined a possible role of ZNF9 in the regulation of translation in DM2 patients. We have found that ZNF9 interacts with the 5′ UTRs of TOP (terminal oligopyrimidine tract) mRNAs encoding human ribosomal protein, RPS17, poly(A)-binding protein, PABP1, and elongation factors, eEF1A and eEF2. The binding activity of ZNF9 toward these TOP-containing 5′ UTRs is reduced in DM2 muscle. Consistent with the reduction of this activity, the levels of RPS17, PABP, eEF1A and eEF2 proteins are also diminished in DM2 muscle. The reduction of ZNF9 RNA-binding activity in DM2 correlates with a decrease of ZNF9 protein levels in cytoplasm of DM2 muscle cells. We have found that the reduction of ZNF9 is caused by expression of the mutant CCUG repeats. This decrease of proteins of translational apparatus in DM2 correlates with a reduction of a rate of protein synthesis in myoblasts from DM2 patients. We found that the ectopic expression of ZNF9 in DM2 myoblasts corrects rate of protein synthesis suggesting that the alterations in CCUG-ZNF9-TOP mRNAs pathway are responsible for the reduction of the rate of protein translation in DM2 muscle cells.

Myotonic dystrophy 2 (DM2) is a multisystem skeletal muscle disease caused by an expansion of tetranucleotide CCTG repeats, the transcription of which results in the accumulation of untranslated CCUG RNA. In this study, we report that CCUG repeats both bind to and misregulate the biological functions of cytoplasmic multiprotein complexes. Two CCUG-interacting complexes were subsequently purified and analyzed. A major component of one of the complexes was found to be the 20S catalytic core complex of the proteasome. The second complex was found to contain CUG triplet repeat RNA-binding protein 1 (CUGBP1) and the translation initiation factor eIF2. Consistent with the biological functions of the 20S proteasome and the CUGBP1-eIF2 complexes, the stability of short-lived proteins and the levels of the translational targets of CUGBP1 were shown to be elevated in DM2 myoblasts. We found that the overexpression of CCUG repeats in human myoblasts from unaffected patients, in C2C12 myoblasts, and in a DM2 mouse model alters protein translation and degradation, similar to the alterations observed in DM2 patients. Taken together, these findings show that RNA CCUG repeats misregulate protein turnover on both the levels of translation and proteasome-mediated protein degradation.

The epigenetic silencing of exogenous transcriptional units integrated into the genome represents a critical problem both for long-term gene therapy efficacy and for the eradication of latent viral infections. We report here that limitation of essential amino acids, such as methionine and cysteine, causes selective up-regulation of exogenous transgene expression in mammalian cells. Prolonged amino acid deprivation led to significant and reversible increase in the expression levels of stably integrated transgenes transcribed by means of viral or human promoters in HeLa cells. This phenomenon was mediated by epigenetic chromatin modifications, because histone deacetylase (HDAC) inhibitors reproduced starvation-induced transgene up-regulation, and transcriptome analysis, ChIP, and pharmacological and RNAi approaches revealed that a specific class II HDAC, namely HDAC4, plays a critical role in maintaining the silencing of exogenous transgenes. This mechanism was also operational in cells chronically infected with HIV-1, the etiological agent of AIDS, in a latency state. Indeed, both amino acid starvation and pharmacological inhibition of HDAC4 promoted reactivation of HIV-1 transcription and reverse transcriptase activity production in HDAC4 + ACH-2 T-lymphocytic cells but not in HDAC4 − U1 promonocytic cells. Thus, amino acid deprivation leads to transcriptional derepression of silenced transgenes, including integrated plasmids and retroviruses, by a process involving inactivation or down-regulation of HDAC4. These findings suggest that selective targeting of HDAC4 might represent a unique strategy for modulating the expression of therapeutic viral vectors, as well as that of integrated HIV-1 proviruses in latent reservoirs without significant cytotoxicity.