Repeat-associated non-AUG translation induces cytoplasmic aggregation of CAG repeat-containing RNAs

Abstract

CAG trinucleotide repeat expansions cause several neurodegenerative diseases, including Huntington's disease and spinocerebellar ataxia. RNAs with expanded CAG repeats contribute to disease in two unusual ways. First, these repeat-containing RNAs may agglomerate in the nucleus as foci that sequester several RNA-binding proteins. Second, these RNAs may undergo aberrant repeat-associated non-AUG (RAN) translation in multiple frames and produce aggregation-prone proteins. The relationship between RAN translation and RNA foci, and their relative contributions to cellular dysfunction, are unclear. Here, we show that CAG repeat-containing RNAs that undergo RAN translation first accumulate at nuclear foci and, over time, are exported to the cytoplasm. In the cytoplasm, these RNAs are initially dispersed but, upon RAN translation, aggregate with the RAN translation products. These RNA–RAN protein agglomerates sequester various RNA-binding proteins and are associated with the disruption of nucleocytoplasmic transport and cell death. In contrast, RNA accumulation at nuclear foci alone does not produce discernable defects in nucleocytoplasmic transport or cell viability. Inhibition of RAN translation prevents cytoplasmic RNA aggregation and alleviates cell toxicity. Our findings demonstrate that RAN translation-induced RNA-protein aggregation correlates with the key pathological hallmarks observed in disease and suggest that cytoplasmic RNA aggregation may be an underappreciated phenomenon in CAG trinucleotide repeat expansion disorders.

CAG repeat expansions create splicing acceptor sites and produce aberrant repeat-containing RNAs

“…A. Schematic for constructs with 240×CAG repeats with a variable 250-base flanking sequence 12 . Some flanking sequences result in retention of the repeat-containing RNA in the nucleus while others induce RAN translation and cell toxicity.…”

“…Four 10-cm dishes expressing CAG RAN fused to BFP at the C-terminus 12 , downstream of the repeat tract, were induced with doxycycline. After 24 hours, the cells were washed once with DPBS and then lysed with 1 mL of RIPA buffer supplemented with HALT protease and phosphatase inhibitors and DTT.…”

“…In our earlier work, we showed that RNAs that consist primarily of expanded CAG trinucleotide repeats, which are not present in a canonical open reading frame (ORF), accumulate at nuclear foci. These repeat-containing RNAs did not undergo RAN translation even when the repeat number was increased to >400 tandem CAGs 5,12 . RAN translation is reported to be influenced by the sequences adjacent to the repeat tract 10,13 .…”

“…To assess the role of flanking sequences, we previously generated a library of CAG repeat-containing plasmids where various 250 nucleotide long sequences were cloned upstream of 240×CAG repeats 12 . We found that a subset of these flanking sequences led to RAN translation of the CAG repeat, while others were still retained at nuclear foci 12 . We were not able to identify sequence motifs in the upstream flanking sequences that differentiated the two classes.…”

“…Our hypothesis was guided by three observations. One, livecell RNA imaging experiments showed that RNAs that undergo RAN translation are first retained at nuclear foci for a prolonged period where they colocalize with the splicing machinery 12 . Two, mammalian 3’ splice sites have a nearly invariant ‘AG’ dinucleotide that marks the splice junction (minimal splice acceptor represented as Y 6-12 NAG NNN , where Y 6-12 indicates the upstream pyrimidine rich tract, N is any nucleotide, and the bold bases mark the exon) 14 .…”