Insight into the degradation pathway of proteins produced by ribosomes stalled at the termination codon

Protein translation is an essential cellular process that must be efficient, accurate and appropriately regulated to guarantee the correct homeostasis and cell survival. When ribosomes fail to complete protein translation, cells developed mechanisms of quality control that target to degradation both the incomplete polypeptides and the mRNAs. However, more studies are necessary to understand how products originated from ribosome stalled at the ER membrane are recognised and targeted to degradation. We found that imposing a conventional termination at the C‐terminus of the Foot‐and‐Mouth Disease Virus (FMDV) peptide 2A induced translational stalling at the termination codon of both free and ER‐bound mammalian ribosomes. Reporter proteins tagged at their C‐terminus with this 2A sequence were highly less expressed in living cells, due to reduced rate of synthesis and proteasomal degradation. In this PhD project, we exploited the 2A stalling mechanism in mammalian cells to investigate the degradation pathway of polypeptides translated by stalled ER‐bound ribosomes. We found that ER‐targeted 2A‐stalled proteins are inserted in the ER lumen and recognised by the pivotal ER‐resident chaperon BiP/GRP78. We hypothesised that BiP/GRP78 binding to peptides derived from stalling ribosomes is responsible of their targeting to degradation mainly via the ER‐associated degradation (ERAD) pathway, as in the case of misfolded proteins. In fact, our findings strongly indicated that 2A proteins derived by ER‐bound stalled ribosomes are retro‐translocated from the ER lumen to the cytosol and processed by the AAA ATPase VCP/p97 and its associated deubiquitinase YOD1 before their proteasomal degradation. Interestingly, we found that also the ribosome-associated quality control E3 ubiquitin ligase Listerin is involved in 2A polypeptides polyubiquitination and degradation, suggesting a crosstalk between two different protein quality control systems at the ER membrane in case of ribosomes stalling of translation. Surprisingly, we obtained indications that the products derived from ER‐stalled ribosomes could be not misfolded or unfolded, suggesting that this multiple ER associated quality control system is able to specifically discriminate the products of stalled ribosome from those proteins normally translated with a mechanism different from canonical ERAD, in which the ribosome‐associated quality control complex may also have an implication. In the present study, we identified two C‐terminal peptide sequences in the human proteome sharing the same three C‐terminal amino acids of 2A, which similarly cause stalling at the STOP‐codon and degradation of the associated protein by the ubiquitin-proteasome pathway. These results suggest that the termination codon‐mediated stalling could affect a broad range of polypeptide sequences, representing a novel post-translational mechanism to control protein expression. A C‐terminal sequence diverse from 2A, known to induce stall of ribosomes in the 3’ UTR of the AMD1 mRNA, was also tested and revealed instead a mechanism of stalling different from the one induced by 2A.