RAN Translation Regulated by Muscleblind Proteins in Myotonic Dystrophy Type 2

Abstract

SUMMARY Several microsatellite-expansion diseases are characterized by the accumulation of RNA foci and RAN proteins raising the possibility of a mechanistic connection. We explored this question using myotonic dystrophy type 2, a multisystemic disease thought to be primarily caused by RNA gain-of-function effects. We demonstrate the DM2 CCTG•CAGG expansion expresses sense and antisense tetrapeptide poly-(LPAC) and poly-(QAGR) RAN proteins, respectively. In DM2 autopsy brains, LPAC is found in neurons, astrocytes and glia in grey matter and antisense QAGR proteins accumulate within white matter. LPAC and QAGR proteins are toxic to cells independent of RNA gain of function. RNA foci and nuclear sequestration of CCUG transcripts by MBNL1 is inversely correlated with LPAC expression. These data suggest a model, that involves nuclear retention of expansion RNAs by RNA-binding proteins (RBPs) and an acute phase in which expansion RNAs exceed RBP sequestration capacity, are exported to the cytoplasm and undergo RAN translation.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

“…The CTG, CCTG, and G 4 C 2 repeats associated with DM1, DM2, and C9ALS/FTD, respectively, undergo repeat-associated non-ATG (RAN) translation (Zu et al, 2011, Zu et al, 2013) (Zu et al, 2017). We hypothesized that dCas9-mediated transcriptional repression would also yield reduced RAN protein abundance.…”

“…HEK293Tcells were transfected with one of the following plasmids that express tagged RAN translated products: pcDNA-6XStop-(CTG) 150 -3X(FLAG-HA-cMyc-His), pcDNA-6XStop-(CCTG) 137 -3X(FLAG-HA-cMyc-His) or pcDNA-6XStop-(G 4 C 2 ) 120 -3X(FLAG-HA-cMyc-His) (Zu et al, 2011, Zu et al, 2013, Zu et al, 2017). These cells were co-transfected with the pXdCas9 plasmid expressing the dCas9 protein (Cheng et al, 2013), and U6 expression vectors (Mali et al, 2013) expressing the control gRNA or gRNA's targeting either strand of the CTG, CCTG and G 4 C 2 repeats ((CAG) 6 or (CUG) 6 gRNAs, (CAGG) 5 , (AGGC) 5 , (GCAG) 5 ,or (CCUG) 5 gRNAs and (C 4 G 2 ) 3 or (G 4 C 2 ) 3 gRNAs, respectively).…”

Regulation of mRNA Translation in Neurons—A Matter of Life and Death

“…However, recent evidence demonstrates that repeat length constitutes a major determinant influencing translation. While repeat length is often positively correlated with the levels of both AUG-initiated and RAN translation (Krans et al, 2016; Krauss et al, 2013; Scoles et al, 2015; Zu et al, 2011, 2013, 2017), RAN translation is only observed from transcripts with pathogenic repeats (Gaspar et al, 2000; Mori et al, 2013a; Todd et al, 2013; Zu et al, 2011, 2013). A similar dependence on pathogenic repeat length has been observed for frameshifting (Saffert et al, 2016).…”

“…C9ORF72 ALS patients experience upper and lower motor neuron death while C9ORF72 FTD patients primarily lose neurons in the frontal and temporal cortices, yet the two sets of patients have indistinguishable distributions of dipeptide repeats (Mackenzie et al, 2013). However, in contrast, aggregates of polyLeu-Pro-Ala-Cys in the brains of patients with myotonic dystrophy type 2 appear specifically in the regions with neuronal death (Zu et al, 2017). …”

“…This problem can be addressed by experimentally altering the ratio of mutant transcript to protein. The toxicity of RAN products has been demonstrated directly by increasing the ratio of protein to RNA through exogenous application of synthetic peptides, addition of an upstream AUG to increase translation from an mRNA, or use of alternative codons that do not form RNA hairpins to encode the repetitive protein (Boeynaems et al, 2016; Freibaum et al, 2015; Kwon et al, 2014; Lee et al, 2016; May et al, 2014; Mizielinska et al, 2014; Wen et al, 2014; Yamakawa et al, 2015; Zhang et al, 2014b; Zu et al, 2017, 2013). These approaches demonstrated the toxicity of arginine-containing C9ORF72 RAN products.…”