Hexanucleotide repeat expansion mutations in C9ORF72 cause amyotrophic lateral sclerosis and frontotemporal dementia. A key feature of the mutation is the abnormal expression of 6 different dipolypeptide repeat proteins (DPRs) which accumulate in the brain. Two of these DPRs comprise Gly-Arg and Pro-Arg repeats and are particularly toxic to cell and animal models. Recently we reported that 101x repeats of arginine-rich DPRs are potent stallers of translation, but the mechanisms remain undetermined (Radwan, Ang et al. 2019).
Ribosomal stalling is known to be important as part of ribosome-associated quality control and can be regulated by a number of genes which are proposed to play a role in controlling translation rates and in triggering degradation of aberrant mRNA and stalled polypeptide. We hypothesize that in the case of polybasic protein sequences, such as Arg-rich DPRs, the positively charge polymer can electrostatically block the negatively charged ribosome exit tunnel. However, recent studies have shown significant translational arrest on polyadenylate sequences, which encode polyLys, but not on polyLys encoded by mixed lysine codon sequences. In addition, stalling on polyadenylate sequences is positively regulated by E3 ubiquitin ligase ZNF598 (Juszkiewicz and Hegde 2017). This suggests that translational arrest during translation of positively charged protein polymers is in part regulated, rather than purely affected by electrostatic jamming.
We have begun to investigate the mechanisms that mediate stalling by Pro-Arg and Gly-Arg and whether they are the same as that for polyLys. We have found that while knockdown of ZNF598 alleviates stalling of polyadenylate-mediated polyLys sequences it has no effect in case of 101x Gly-Arg and Pro-Arg indicating that stalling during translation of Arg-rich DPRs involves distinct mechanisms. Our ongoing work is to apply a genome-wide CRISPR knockout screen to find positive and negative regulators of ribosomal stalling.