Ribosome stalling occurs on defective mRNAs and often triggers ribosome-associated quality control (RQC) machinery that leads to ribosome recycling as well as degradation of both mRNA and aberrant polypeptide. The known mechanisms of RQC come largely from studies of mRNA lacking stop codons, whereby ribosomes become stalled at the polyadenylate sequence in the 3`UTR. We previously reported that long arginine-rich dipeptide repeats (DPR), mimicking abnormal proteins expressed in C9orf72-mediated amyotrophic lateral sclerosis, also caused translation arrest but in RNA-independent manner (Radwan et al. 2020). What causes this stalling and whether these mechanisms are similar to canonical mRNA-mediated arrest remained unclear.
In this work, we found that the readthrough efficiency of arginine-rich DPRs drops with the increase in peptide length, and only DPRs longer than 40 repeats cause significant translation arrest. We also found how different amino acid composition affects DPR readthrough: ribosome failed to efficiently synthesize all DPRs enriched in charged amino acids (Arg, Asp, Glu or Lys), though stalling mechanisms differed between positively and negatively charged DPRs. Finally, all genes that are known to facilitate arrest on polyadenylate sequence, such as ZNF598 and members of RQC-trigger complex, do not affect the readthrough of arginine-rich DPRs. Collectively our findings indicate that the mechanisms involved in stalling on defective mRNAs and stalling caused by long charged DPRs are unequivocally distinct. Our working model posits that emergent, nascent DPRs either become jammed in the ribosome exit tunnel and/or collapse the translational machinery by sequestering them into non-functional aggregates.