In amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) caused by C9ORF72 hexanucleotide expansions, five types of aberrant dipeptide repeat (DPR) proteins are expressed and accumulate in patient cells. Two of the five are arginine-rich and both are selectively toxic to cell culture and animal models suggesting the arginine composition mediates a specific link to disease pathogenesis. We recently showed that Arg-rich DPRs severely stall ribosomes during their translation (Radwan et al. 2020) and hypothesized that this arose through an electrostatic blocking mechanism of emergent Arg-rich nascent chains with the negatively charged ribosome exit tunnel.
In this study, we explored the effect of Arg-rich DPRs in more detail and how it compared mechanistically to canonical polyadenylate-mediated stalling. Genome wide CRISPR knockout screens for modifiers of stalling yielded 100 candidate genes that either increase stalling or facilitate read-through on polyadenylate mRNA sequence. This list included genes that are already known to regulate polyadenylate-mediated stalling, which validated the approach. Apart of that, we identified 47 potential regulators of stalling on Arg-rich DPRs and, notably, there was a little overlap between two groups: only 28 genes modified translational arrest with both stallers. Moreover, knockouts of ZNF598 and RACK1 resulted in read-through of polyadenylate sequence, but caused even more stalling with Arg-rich DPRs. More generally, we observed little evidence of any alleviation of Arg-rich DPR-mediated stalling by gene knockout. These findings point to a divergence in the mechanistic underpinning of stalling between polyadenylate sequence versus Arg-rich DPRs. It appears that Arg-rich DPRs cause a more nonrecoverable stall which cannot be efficiently cleared by cellular quality control machineries. We postulate that ability of long Arg-rich nascent chains to electrostatically jam the ribosome and to sequester other components of translation machinery into aggregates explains why synthesis of these pathological repeats leads to severe translational arrest.