Double-stranded RNA is pathogenic in Drosophila models of expanded repeat neurodegenerative diseases

Abstract

The pathogenic agent responsible for the expanded repeat diseases, a group of neurodegenerative diseases that includes Huntington's disease is not yet fully understood. Expanded polyglutamine (polyQ) is thought to be the toxic agent in certain cases, however, not all expanded repeat disease genes can encode a polyQ sequence. Since a repeat-containing RNA intermediary is common to all of these diseases, hairpin-forming single-stranded RNA has been investigated as a potential common pathogenic agent. More recently, it has become apparent that most of the expanded repeat disease loci have transcription occurring from both strands, raising the possibility that the complementary repeat RNAs could form a double-stranded structure. In our investigation using Drosophila models of these diseases, we identified a fortuitous integration event that models bidirectional repeat RNA transcription with the resultant flies exhibiting inducible pathology. We therefore established further lines of Drosophila expressing independent complementary repeat RNAs and found that these are toxic. The Dicer pathway is essential for this toxicity and in neuronal cells accounts for metabolism of the high copy number (CAG.CUG)(100) double-stranded RNAs down to (CAG)(7) single-stranded small RNAs. We also observe significant changes to the microRNA profile in neurons. These data identify a novel pathway through which double-stranded repeat RNA is toxic and capable of eliciting symptoms common to neurodegenerative human diseases resulting from dominantly inherited expanded repeats.

Drosophila Pur-α binds to trinucleotide-repeat containing cellular RNAs and translocates to the early oocyte

“…38 While Pur-α associates with RNAi factors (Fig. S1B), it is dispensable for RNAi and miRNA-mediated silencing (data not shown).…”

Targeting CAG repeat RNAs reduces Huntington’s disease phenotype independently of huntingtin levels

“…Dicer activity on the hairpin-like double-stranded structure in expanded CAG repeats of HTT-e1 results in the biogenesis of small CAG-repeated RNAs (sCAGs) (26) with toxic activity (9). In addition, binding of dicer to expanded CAG repeats has been proposed to contribute to abnormal miR expression profiles that may underlie pathogenic alterations in gene expression (27). To evaluate whether the detrimental effect of sRNAs was reversed by LNA-CTG, we isolated an sRNA-enriched fraction (<200 nt) from the striatum of WT and R6/2 mice injected with LNA-SCB and from R6/2 mice injected with LNA-CTG (pooled extracts from at least 11 mice per condition) and determined cell death after transfection into a differentiated neuronal cell line ( Figure 5A).…”

“…Expanded CAG repeats form a hairpin-like secondary structure that causes RNA toxicity through diverse mechanisms involving aberrant interactions with proteins and consequent altered functions (12). These include dicer and nucleolin (NCL), which underlie deleterious sRNA expression profiles (9,27) and decreased Rn45s expression levels mediating nucleolar stress (5, 28), respectively.…”

Small interfering RNAs based on huntingtin trinucleotide repeats are highly toxic to cancer cells

“…Expression of the two transcripts led to the generation of Dicer-2 (dcr-2) and ago2-dependent 21-nt TNR-derived siRNAs resulting in high toxicity to the cells. In a separate study, it was shown that expression of these complementary repeat RNAs leads to dcr-2-dependent neurodegeneration [28]. These results suggest that co-expression of CAG and CUG repeat-derived sequences may dramatically enhance toxicity in human repeat expansion diseases in which anti-sense transcription occurs.…”