Expression of RNA CCUG Repeats Dysregulates Translation and Degradation of Proteins in Myotonic Dystrophy 2 Patients

Abstract

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.

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

“…CUGBP1 binds to CUG repeats as a single protein; however, it binds to CCUG repeats as a component of multiprotein complexes. 18,32 Consistent with elevation of CUGBP1, translation of CUGBP1 targets is altered by CUG and CCUG repeats after CUGBP1 increase.…”

“…However, recent data suggest that high levels of short repeats might also be toxic. 24,32 In support of the critical role of high levels of short repeats, our data show that large amounts of short CUG and CCUG repeats cause toxic events such as foci accumulation, elevation of CUGBP1, and reduction of ZNF9. We suggest that the foci formation and elevation of CUGBP1 in CUG 25 and CCUG 36 -inducible cells is caused by the cumulative effect of high levels of CUG and CCUG repeats of normal length.…”

“…At 24 to 48 hours after the transcription pulse, CCUG 100 RNA completely reduced ZNF9, whereas CCUG 36 could not completely decrease ZNF9 levels ( Figure 10, A and B). Because CCUG repeats increase the stability of a cdk inhibitor, p21, through the inhibition of the 20S proteasome, 32 we determined the levels of p21 after a single pulse of transcription of CCUG repeats, finding that both short and long CCUG repeats increased p21, but that this increase took place at later time points of accumulation of CCUG repeats ( Figure 10, A and B). Based on these data, we conclude that the increase of p21 levels is a late event of CCUG accumulation.…”

“…9,15,16,18,24,32,34,35 CUG and CCUG repeats aggregate in DM1 and DM2 patients; however, it remains unknown whether these foci are formed by long, nondegraded mutant CUG and CCUG RNAs or by short products of degradation of RNA expansions. 9,15,16,32,34,35 It is also not known whether the formation of these aggregates is responsible for all key molecular abnormalities in DM cells, besides sequestration of MBNL1. 16,21,35 In fact, several reports indicate the lack of correlation between foci formation and severity of disease.…”

“…29,30 In addition, protein expression is altered in DM2 patients at the level of protein degradation through the inhibition of the proteasome-ubiquitin pathway. 32,33 The expansion of CUG and CCUG repeats changes biological activities of several RNA binding proteins including muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). 11-14,16,18 -26,32 Whereas some mutant CUG and CCUG repeats form aggregates that sequester MBNL1, other CUG/CCUG repeats remain soluble, stabilizing CUGBP1 and leading to an elevated amounts of CUGBP1.…”