Repeat Associated Non-AUG Translation (RAN Translation) Dependent on Sequence Downstream of the ATXN2 CAG Repeat

Abstract

Spinocerebellar ataxia type 2 (SCA2) is a progressive autosomal dominant disorder caused by the expansion of a CAG tract in the ATXN2 gene. The SCA2 disease phenotype is characterized by cerebellar atrophy, gait ataxia, and slow saccades. ATXN2 mutation causes gains of toxic and normal functions of the ATXN2 gene product, ataxin-2, and abnormally slow Purkinje cell firing frequency. Previously we investigated features of ATXN2 controlling expression and noted expression differences for ATXN2 constructs with varying CAG lengths, suggestive of repeat associated non-AUG translation (RAN translation). To determine whether RAN translation occurs for ATXN2 we assembled various ATXN2 constructs with ATXN2 tagged by luciferase, HA or FLAG tags, driven by the CMV promoter or the ATXN2 promoter. Luciferase expression from ATXN2-luciferase constructs lacking the ATXN2 start codon was weak vs AUG translation, regardless of promoter type, and did not increase with longer CAG repeat lengths. RAN translation was detected on western blots by the anti-polyglutamine antibody 1C2 for constructs driven by the CMV promoter but not the ATXN2 promoter, and was weaker than AUG translation. Strong RAN translation was also observed when driving the ATXN2 sequence with the CMV promoter with ATXN2 sequence downstream of the CAG repeat truncated to 18 bp in the polyglutamine frame but not in the polyserine or polyalanine frames. Our data demonstrate that ATXN2 RAN translation is weak compared to AUG translation and is dependent on ATXN2 sequences flanking the CAG repeat.

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).…”

“…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.…”

“…In the absence of a near-AUG, translation of FMR1polyGly does not occur. In addition to regulation by upstream sequences, sequence downstream of repeats may also influence levels of RAN translation (Scoles et al, 2015; Zu et al, 2011). …”

Motor Dysfunctions and Neuropathology in Mouse Models of Spinocerebellar Ataxia Type 2: A Comprehensive Review

“…This work has caused a turmoil in the field of DNA repeat expansions disorders, with new non-ATG proteins being reported in several neurodegenerative traits, rendering the field far more complex than it was first considered (Cleary and Ranum, 2014). In SCA2, this phenomenon was also observed for the ATXN2 gene, although in lower levels compared to canonical ATG translation and the study failed to detect polyserine and polyalanine proteins (Scoles et al, 2015). …”

RNA biology of disease-associated microsatellite repeat expansions

“…Repeat expansion diseases where this mechanism has been observed now include SCA2, SCA8, SCA31, HD, FXTAS/FXPOI, and C9FTD/ALS [9, 13, 27, 96, 129, 218, 261, 290, 339, 340]. RAN translation of xtrRNA sequence can occur in many contexts, including repeat expansions found in untranslated regions, retained introns, and even those embedded in coding exons [96].…”