DDX6 regulates sequestered nuclear CUG-expanded DMPK-mRNA in dystrophia myotonica type 1.
Bottom Line: DDX6 overexpression relieves DM1 mis-splicing, and causes a significant reduction in nuclear DMPK-mRNA foci.Conversely, knockdown of endogenous DDX6 leads to a significant increase in DMPK-mRNA foci count and to increased sequestration of MBNL1 in the nucleus.Finally, we show that DDX6 unwinds CUG-repeat duplexes in vitro in an adenosinetriphosphate-dependent manner, suggesting that DDX6 can remodel and release nuclear DMPK messenger ribonucleoprotein foci, leading to normalization of pathogenic alternative splicing events.
Affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, Building 1240, DK-8000 Aarhus C, Denmark.Show MeSH
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Mentions: Since DDX6 overexpression reduced the nuclear CUG-foci formation, we speculated that this potentially could reduce the reported mis-splicing events, which are hallmarks of DM1. To test this, we utilized RNA purified from either GFP- or DDX6-transduced cells and performed semi-quantitative hot RT-PCR and quantified the level of mis-splicing of mRNAs encoding human IR2, GNAS, PPP2R5C, SPAG9 and NFIX (10). We observed a modest but significant defect in splicing of IR2 (P < 0.01) and ppp2r5c (P < 0.05) pre-mRNA in DM1 fibroblasts compared to untreated WT fibroblasts (Figure 4A and B, compare lanes 5–8 and lanes 3–4, respectively). Most importantly, fibroblasts expressing FLAG-DDX6 allowed for a partial rescue of the IR2 (P < 0.01) and ppp2r5c mis-splicing events (P < 0.05), suggesting that the decrease in nuclear CUG-foci translates into functional relief of DM1 specific mis-splicing, whereas splicing in WT cells remained unaffected. The defective IR2 splicing phenotype in DM1 cells and the restoration to ‘near-normal’ splicing pattern by DDX6 overexpression was confirmed by qRT-PCR, by which we quantified the individual transcripts either containing or lacking alternative exon 11 (Supplementary Figures S7A and B). Deregulation of ppp2r5c alternative splicing in DM1 cells was modest but similar to previously published phenotypes (10). Alternative splicing of pre-mRNAs encoding GNAS, SPAG9 or NFIX was not significantly changed between WT and DM1 fibroblasts (unpublished observations) in contrast to previous reports using DM1 muscle biopsies (10). This was not an effect of non-quantitative PCR conditions as evidenced by the linear range of amplification observed for the IR2 (Figure 4A lanes 1–4) and by qRT-PCR (Supplementary Figure S7). This is consistent with previous reports, stating that DMPK-mRNA expression in undifferentiated fibroblasts is much lower than that of differentiated muscle cells (21), which suggests that robust mis-splicing is evident only in cells of higher CUG-mRNA levels. Although we cannot formally exclude the possibility that higher DDX6 expression may change the mRNA stability of specific IR2/pp2r5c splice-variants, our data indicates that DDX6 overexpression partially relieves DM1 specific mis-splicing of IR2 pre-mRNA in human DM1 fibroblasts.
Affiliation: Department of Biomedicine, Aarhus University, Wilhelm Meyers Allé 4, Building 1240, DK-8000 Aarhus C, Denmark.