Repeat‐associated non‐AUG translation from antisense CCG repeats in fragile X tremor/ataxia syndrome

Abstract

Objective Repeat associated non-AUG (RAN) translation drives production of toxic proteins from pathogenic repeat sequences in multiple untreatable neurodegenerative disorders. Fragile X-associated tremor/ataxia syndrome (FXTAS) is one such condition, resulting from a CGG trinucleotide repeat expansion in the 5′ leader sequence of the FMR1 gene. RAN proteins from the CGG repeat accumulate in ubiquitinated inclusions in FXTAS patient brains and elicit toxicity. In addition to the CGG repeat, an antisense mRNA containing a CCG repeat is also transcribed from the FMR1 locus. We evaluated whether this antisense CCG repeat supports RAN translation and contributes to pathology in FXTAS patients. Methods We generated a series of CCG RAN translation specific reporters and utilized them to measure RAN translation from CCG repeats in multiple reading frames in transfected cells. We also developed antibodies against predicted CCG RAN proteins and used immunohistochemistry and immunofluorescence on FXTAS patient tissues to measure their accumulation and distribution. Results RAN translation from CCG repeats is supported in all three potential reading frames, generating polyproline, polyarginine, and polyalanine proteins, respectively. Their production occurs whether or not the natural AUG start upstream of the repeat in the proline reading frame is present. All three frames show greater translation at larger repeat sizes. Antibodies targeted to the antisense FMR polyproline and polyalanine proteins selectively stain nuclear and cytoplasmic aggregates in FXTAS patients and colocalize with ubiquitinated neuronal inclusions. Interpretation RAN translation from antisense CCG repeats generates novel proteins that accumulate in ubiquitinated inclusions in FXTAS patients.

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

“…This discovery opened the floodgates, and soon evidence accumulated for RAN translation of CAG and other repeats in additional contexts. To date, multiple repeat-encoded proteins that are translated using non-canonical initiation sites have been identified in aggregates in patients with repeat-expansion diseases, and many other repetitive proteins can be generated from RAN translation in vitro (Table 1; (Krans et al, 2016; Todd et al, 2013; Zu et al, 2011, 2013). For example, six RAN proteins, corresponding to all frames of sense and antisense transcripts, are produced from the hexanucleotide expansion found in an intron of C9ORF72, the most common familial cause of ALS and frontotemporal dementia (FTD) (Zu et al, 2013).…”

“…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).…”

“…A similar dependence on pathogenic repeat length has been observed for frameshifting (Saffert et al, 2016). However, for other RAN-translated or frameshifted proteins, the relationship between repeat length and protein levels is less clear (Kearse et al, 2016; Krans et al, 2016; Todd et al, 2013; Toulouse et al, 2005; Zu et al, 2011). The positive correlation between repeat length and non-canonical translation levels may be due to the tendency of longer repeats to form secondary structures.…”

“…While poly(CAG) RAN translation was supported by a series of constructs with similar repeats but with unique upstream flanking sequences, the abundance and size of the RAN products varied, indicating translation initiates at multiple sites (Todd et al, 2013; Zu et al, 2011). In experiments comparing transcripts differing only in the presence or absence of an in-frame AUG codon upstream of the repeat, the presence of an AUG usually, but not always, resulted in greater translation, suggesting that some upstream sequences may influence start site selection by simply providing an in-frame AUG start codon (Bañez-Coronel et al, 2015; Kearse et al, 2016; Krans et al, 2016; Scoles et al, 2015; Todd et al, 2013; Zu et al, 2011, 2013). For RAN products that do not initiate within the repeat, upstream sequences are essential.…”

“…If all RAN translation were dependent upon scanning, the presence of an upstream AUG in a different frame would be predicted to diminish or prevent RAN translation. However, translation of most RAN products is unaffected by out-of-frame upstream AUGs (Bañez-Coronel et al, 2015; Krans et al, 2016; Zu et al, 2011, 2013). Furthermore, canonical cap-dependent translation is unlikely to be involved in the translation of repeats in introns or antisense transcripts.…”