Alternative Splicing of MBNL1 and MBNL2 Regulates the Nucleocytoplasmic Distribution of CUG RNA Foci in DM1 Cell Models

Background/Objectives: Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy. DM1 is caused by a CTG trinucleotide repeat expansion in the 3′-untranslated region of the DMPK gene. Expanded CUG repeat RNA forms nuclear CUG RNA foci that sequester the RNA-binding protein MBNL1, leading to loss of MBNL1 function and resulting in splicing abnormalities. MBNL1 autoregulates its nuclear localization by repressing the inclusion of exon 5, which encodes part of its nuclear localization signal. In DM1, exon 5 inclusion is increased due to loss of MBNL1 function. However, whether inclusion of exon 5 contributes to the formation of nuclear CUG RNA foci remains unclear; we investigated this relationship in the present study. Methods: We used dCasRx, the catalytically inactive form of Cas13d, together with guide RNAs targeting sequences within and around exon 5 to identify an optimal sequence for exon 5 skipping. An antisense oligonucleotide (ASO) designated ex5-ASO, designed from the selected sequence, was transfected into a cellular DM1 model and DM1 patient-derived fibroblasts to assess their effects on RNA foci. Results: ex5-ASO successfully induced skipping of not only MBNL1 exon 5 but also MBNL2 exon 5 in a dose-dependent manner. The cytoplasmic fractions of both MBNL1 and MBNL2 increased following ex5-ASO transfection. Remarkably, ex5-ASO treatment reduced nuclear RNA foci and induced the appearance of cytoplasmic foci. However, despite reduced nuclear RNA foci and substantial residual nuclear MBNL1/2, mis-splicing persisted after ex5-ASO treatment. Conclusions: The level of MBNL1/2 exon 5 inclusion affects nuclear RNA foci formation, suggesting that the increased exon 5 inclusion observed in DM1 is not merely a consequence of MBNL loss of function but may also contribute to the nuclear accumulation of RNA foci and sequestration of MBNL1/2.