Regulation of Toxic RNA Foci and Repeat Expansion DMPK Transcripts: Role of MBNL Proteins and RNA Decay Pathways

Myotonic dystrophy type 1 (DM1) is a progressive, multisystemic disorder caused by an expansion of CTG repeats in the 3 untranslated region of the DMPK gene. When transcribed the mutant RNAs accumulate in affected tissues appearing as distinct foci when visualised by in situ hybridisation. The RNA foci are aggregates of CUG repeat-containing RNAs that sequester RNA-binding proteins, particularly muscleblind-like (MBNL) proteins, leading to their dysfunction and causing downstream molecular and cellular defects. Here we show the double knock-out of MBNL1 and 2 prevents RNA foci formation and nuclear retention of mutant DMPK mRNA in DM1 cells as well as promoting their degradation and nuclear export. Using stochastic optical reconstruction microscopy (STORM), we find the presence of both large foci and micro foci in DM1 cells. Large foci consist of multiple DMPK transcripts, while many micro foci are (CUG)n fragments. The absence of MBNL proteins not only prevents the aggregation of multiple DMPK transcripts into large foci, but also promotes their degradation and nuclear processing. However, although a substantial amount of MBNL1 proteins are bound to the mutant transcripts, the pools of free MBNL1 proteins are similar in DM1 nuclei to those in controls. Furthermore, we have identified several factors that are involved in the control of mutant DMPK mRNA turnover, including XRN2, EXOSC10, UPF1 and STAU1. Our study indicates that these factors are implicated in the RNA foci accumulation and the degradation of mutant DMPK mRNA. UPF1 and STAU1 may have additional roles beyond degradation, impacting the nuclear processing of mutant DMPK mRNA. Our study also highlights the critical role of MBNL proteins in regulating mutant DMPK mRNA metabolism: the absence of MBNLs in DM1 appears to expedite the processing of mutant DMPK mRNA mediated by these RNA decay factors. Significance statementOur investigations uncovered valuable data on the RNA foci dynamics in DM1, revealing the intricate mechanisms that underlie their formation, stability, and turnover. Our findings also contributed to delineate the complex pathways involved in the transportation and degradation of the mutant mRNA and provided insights into the critical role played by MBNL proteins in these processes. Studying the degradation mechanism of mutant DMPK mRNA in myotonic dystrophy may provide a foundation for comprehending the mechanisms of RNA degradation in other diseases caused by short tandem repeat (STR) mutations, such as Huntingtons disease, Fragile X syndrome, and several types of ataxia. Additionally, the use of cutting-edge STORM technology can provide a valuable tool for investigating RNA foci in other STR expansion disorders.