Expansion of CUG RNA repeats causes stress and inhibition of translation in myotonic dystrophy 1 (DM1) cells

Abstract

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.

RNA Foci, CUGBP1, and ZNF9 Are the Primary Targets of the Mutant CUG and CCUG Repeats Expanded in Myotonic Dystrophies Type 1 and Type 2

“…The effect of GFP transcription as a possible toxic factor was assessed previously. 38 It was shown that GFP expression did not induce significant changes in ph-eIF2 levels in the selected cells, indicating that the cells were not under stress. 38 2476 Jones et al AJP November 2011, Vol.…”

“…Our previous work with CUG-inducible CHO cells showed that the levels of CUG 914 RNA at 17 to 24 hours after Dox addition are similar to the levels of the mutant DMPK mRNA in human myoblasts from the patient with DM1. 38 Comparison of CUG 914 and CUG 25 expression on the same gel indicated that the efficiency of CUG 25 RNA expression was significantly higher than that of CUG 914 RNA ( Figure 1B). Although the accumulation of CUG 914 was barely visible at 7 hours after Dox addition, the signals of CUG 25 at this time point were very strong.…”

“…38 To generate TRE-GFP-CTG 25 and TRE-GFP-CCTG 36 plasmids, complementary oligonucleotides CTG 25 , CAG 25 , CCTG 36 , and CAGG 36 were synthesized at Invitrogen (Carlsbad, CA), annealed, and cloned into the EcoRV site of a pTRE-Tight-Bl-AcGFP vector. The resulting sequence of CTG and CCTG repeats was verified by sequencing of the plasmids from both ends.…”

“…38 A similar construct for the regulated expression of short CUG repeats (CUG 25 ) was also generated ( Figure 1A). We applied Northern blot analyses to compare the kinetics of CUG 25/914 accumulation in CHO double-stable cells after a single pulse of transcription.…”

“…Previous studies from our laboratory and other laboratories showed that CUGBP1 elevation is an early event of CUG toxicity; however, it remains unknown whether CUG foci formation precedes elevation of CUGBP1. 38,39 It is also important to determine whether elevation of CUGBP1 and the decrease of ZNF9 are primary events of CCUG accumulation or are consequences of the aggregation of CCUG repeats. The elucidation of the primary targets of CUG and CCUG repeats is important for the development of therapy in DM1 and DM2.…”