“Woodchuck post-transcriptional regulatory element induces nuclear"

NIKOLAOS P MASTROYIANNOPOULOS; MARIANA FELDMAN; JAMES B UNEY; MANI S MAHADEVAN; LEONIDAS A PHYLACTOU

Nantes, France

Congreso; Myology 2005; 2005

Introduction : CTG trinucleotide repeat expansions in the 3’ untranslated region (3’ UTR) of the myotonic dystrophy protein kinase (DMPK) gene is responsible for myotonic dystrophy (DM). Mutant DMPK transcripts aggregate in the nucleus and are thought to trigger dominant effects by interacting with RNA binding proteins, which among other things result to the inhibition of muscle cell differentiation. The woodchuck post-transcriptional regulatory element (WPRE) is a cis-acting module that can enhance transgene expression at the post-transcriptional level. It is believed that when inserted downstream of a given cDNA, it may improve gene expression by increasing RNA export. Objectives : To facilitate WPRE-mediated export of trapped mutant DMPK transcripts to the cytoplasm and therefore increase muscle differentiation in DM cells. Methods : A series of constructs were produced expressing the wild type or mutant DMPK 3’ UTR with or without the WPRE sequence. Following transfection of these constructs in mouse C2C12 myoblasts, RNA and protein analysis was carried to detect transcript transport to the cytoplasm and alterations in muscle differentiation, respectively. Results : WPRE stimulated the export of DM transcripts to the cytoplasm of myoblasts, whereas mutant DM transcripts were mainly retained in the nucleus. Moreover, it repaired defective MyoD levels, known to be involved in DM pathology. Finally, the presence of WPRE downstream of the mutant DMPK transcripts caused a significant increase in the myogenic marker, myogenin and a repair of myoblast differentiation, as assessed by myotube formation. The increase in myotube formation due to WPRE was in some cases 7-fold higher than in the mutant cells. Conclusions : This is the first demonstration of a mechanism by which nuclear blocked transcripts can be liberated to the cytoplasm. The exploitation of this powerful method would be beneficial for DM but also for the study of RNA transport mechanisms in mammalian cells.