title: 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.
authors.affiliation: Department of Biochemistry, Université de Montréal, 2900 Edouard-Montpetit, Montréal, QC H3C 3J7, Canada.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Querido
authors.forename: Franck
authors.initials: F
authors.lastname: Gallardo
authors.forename: Mélissa
authors.initials: M
authors.lastname: Beaudoin
authors.forename: Catherine
authors.initials: C
authors.lastname: Ménard
authors.forename: Pascal
authors.initials: P
authors.lastname: Chartrand
mesh_terms: D000818:Animals; D036681:Fluorescence Recovery After Photobleaching / Q000379:methods; D020869:Gene Expression Profiling; D051379:Mice; D009223:Myotonic Dystrophy / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D013269:Stochastic Processes; D014158:Transcription, Genetic; D019680:Trinucleotide Repeat Expansion; D018911:Trinucleotide Repeats
chemical_list: D012333:RNA, Messenger