Stochastic and reversible aggregation of mRNA with expanded CUG-triplet repeats

Abstract

Transcripts containing expanded CNG repeats, which are found in several neuromuscular diseases, are not exported from the nucleus and aggregate as ribonuclear inclusions by an unknown mechanism. Using the MS2-GFP system, which tethers fluorescent proteins to a specific mRNA, we followed the dynamics of single CUG-repeat transcripts and RNA aggregation in living cells. Single transcripts with 145 CUG repeats from the dystrophia myotonica-protein kinase (DMPK) gene had reduced diffusion kinetics compared with transcripts containing only five CUG repeats. Fluorescence recovery after photobleaching (FRAP) experiments showed that CUG-repeat RNAs display a stochastic aggregation behaviour, because individual RNA foci formed at different rates and displayed different recoveries. Spontaneous clustering of CUG-repeat RNAs was also observed, confirming the stochastic aggregation revealed by FRAP. The splicing factor Mbnl1 colocalized with individual CUG-repeat transcripts and its aggregation with RNA foci displayed the same stochastic behaviour as CUG-repeat mRNAs. Moreover, depletion of Mbnl1 by RNAi resulted in decreased aggregation of CUG-repeat transcripts after FRAP, supporting a direct role for Mbnl1 in CUG-rich RNA foci formation. Our data reveal that nuclear CUG-repeat RNA aggregates are labile, constantly forming and disaggregating structures, and that the Mbnl1 splicing factor is directly involved in the aggregation process.