Defining the Properties and Toxicity Mechanisms of Non-Canonical Translation Products Associated with ALS and Huntington’s Disease Using C. elegans

Expanded guanine/cytosine (G/C)-rich nucleotide repeats are the underlying genetic cause of many age-dependent neurodegenerative diseases. An emerging mechanism underlying disease pathology is an unusual type of protein translation called Repeat-Associated non-AUG (RAN) translation. RAN translation requires extended G/Crich repeats and occurs independently of a canonical start codon, allowing translation in all three reading frames. Antisense RNA from G/C-rich repeats also gives rise to RAN products, causing up to six distinct protein products from one repeat expansion. The toxicity of RAN products in vivo is beginning to be explored. We created codon-varied RAN product models for two different repeats: an intronic GGGGCC repeat expansion that is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS), and the CAG repeat expansion that causes Huntington’s disease (HD). In our C. elegans ALS model, we discovered that the arginine-containing dipeptides, proline-arginine (PR) and glycine-arginine (GR), were the most toxic dipeptides. PR and GR exhibited age-dependent toxicity when expressed in multiple cell types, including motor neurons. Both PR and GR exhibited nuclear localization that was necessary for toxicity. An unbiased whole-genome RNAi screen for suppressors of PR toxicity identified twelve genes. Four of the genes were previously identified in PR modifier screens performed in other systems, suggesting mechanisms of PR toxicity are conserved. My studies are the first to model codon-varied CAG RAN polypeptides in a multicellular animal. Every polypeptide, except for polyLeucine (polyLeu), formed immobile protein aggregates at ~38 repeats. Surprisingly, polyLeu was the most toxic HD RAN polypeptide in every tissue studied. A forward mutagenesis screen combined with a candidate RNAi screen identified three genes that suppressed polyLeu toxicity. Two of the genes encoded transmembrane proteins, and the third gene encoded a deubiquitinase enzyme, suggesting that polyLeu toxicity occurs via disrupted folding of transmembrane proteins. Understanding how different RAN products contribute to HD and ALS is vital for developing appropriate treatments for these diseases.