RNA toxicity in non-coding repeat expansion disorders

Abstract

Several neurodegenerative disorders like amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia (SCA) are caused by non-coding nucleotide repeat expansions. Different pathogenic mechanisms may underlie these non-coding repeat expansion disorders. While gain-of-function mechanisms, such as toxicity associated with expression of repeat RNA or toxicity associated with repeat-associated non-ATG (RAN) products, are most frequently connected with these disorders, loss-of-function mechanisms have also been implicated. We review the different pathways that have been linked to non-coding repeat expansion disorders such as C9ORF72-linked ALS/frontotemporal dementia (FTD), myotonic dystrophy, fragile X tremor/ataxia syndrome (FXTAS), SCA, and Huntington's disease-like 2. We discuss modes of RNA toxicity focusing on the identity and the interacting partners of the toxic RNA species. Using the C9ORF72 ALS/FTD paradigm, we further explore the efforts and different methods used to disentangle RNA vs. RAN toxicity. Overall, we conclude that there is ample evidence for a role of RNA toxicity in non-coding repeat expansion diseases.

Keywords: C9ORF72 ALS/FTD; RNA toxicity; non-coding repeat expansion disorders.

Figure 1. Three possible pathogenic mechanisms of non‐coding repeat expansion disorders—example given for C9ORF72 ALS/FTD

First, the repeat expansion might interfere with the normal transcription of the C9ORF72 gene, leading to loss of function of the C9orf72 protein. Second, repeat‐containing mRNAs might bind to various RNA‐binding proteins, hence disturbing their normal function. This is called “RNA toxicity”. Third, the repeat RNA itself might unconventionally be translated into peculiar toxic RAN peptides. This is called “RAN toxicity”.

Figure 2. C9ORF72 gene structure, transcription, and translation

Four potentially pathogenic RNA species can be discerned. (1) At the pre‐mRNA level, transcription of v1 and v3 might stall at the repeat region, resulting in the generation of abortive transcripts. (2) Transcription of the repeat region in the antisense direction generates antisense transcripts. (3) Ineffective splicing of intron 1 in transcripts v1 and v3 might result in intron 1‐retaining transcripts. (4) Effective splicing of intron 1 in transcripts v1 and v3 might generate repeat‐containing spliced‐out intron 1.

Figure 3. Roadmap to prove/disprove RNA toxicity

Arguments pro/contra RNA toxicity can be generated at four levels of the presumed pathogenic cascade of RNA toxicity.

Figure 4. Constructs used to model C9ORF72 gain‐of‐function toxicity

First, codon‐optimized DPR constructs generate DPRs but do not have the potential to inflict RNA toxicity since the lack of a repetitive sequence. Therefore, these constructs allow an easy modeling of DPR toxicity. Second, ATG repeat constructs can theoretically induce RNA toxicity but by default also generate DPRs. Therefore, RNA toxicity cannot be investigated with these constructs. Third, repeat constructs lacking an ATG start codon (i.e., “non‐ATG repeat constructs”) can also give rise to RNA toxicity while DPR generation is uncertain since it needs to rely on RAN translation. Therefore, by assessing the presence of DPRs these constructs can be used to assess RNA toxicity. Fourth, so‐called “RNA only” constructs should theoretically only give rise to RNA toxicity as the repeat sequence is regularly interrupted by stop codons interfering with RAN translation.

Figure 5. Processes possibly disturbed by C9ORF72 RNA toxicity

(1) Compromised function of nucleolin (NCL) might induce nucleolar stress. (2) mRNA might be retained in the nucleus due to repeat RNA‐induced nuclear accumulation of mRNA export proteins like PABPC. (3) Splicing might be disturbed due to compromised function of several splicing factors like HNRNPH. (4) Nucleocytoplasmic transport might be directly disturbed by repeat RNA via RanGAP1 dysfunction. (5) Translation of mRNA might be altered due to compromised function of translational factors like Pur‐alpha and ZFP106. (6) Cytoplasmic RNA transport might be disturbed by compromised function of RNA transport factors like Pur‐alpha. (7) Autophagy might be compromised by dysfunction of Pur‐alpha.