To address aim (i), we targeted ribosomes translating misfolding-prone proteins. Utilizing a combination of proteomics and ribosome profiling approaches, we were able to develop a highly sensitive proteomics approach to identify co-translational interactors. These specific interactors include known factors involved in the ubiquitin-proteasome pathway, as well as several putative E3 ligases and folding chaperones. Importantly, many of the discovered factors are entirely novel. Based on this, we compiled a candidate list of ribosome quality-control factors for further analysis. All candidates’ deletion strains displayed impaired growth phenotype when exposed to various translation stresses. Several candidates were also found to be required for co-translational degradation of several misfolding-prone proteins. In order to reveal the impact on nascent-chains proteolysis in a proteome-wide manner, we developed a method to map N`-terminal peptides integrity. We are combining this with selective ribosome profiling of the candidates.
To address aim (ii), we combined selective ribosome profiling, imaging, and advanced proteomics with all-atoms molecular dynamics. Focusing on the conserved N-terminal acetyltransferases (NATs) family, we revealed diverging co-translational assembly pathways, where highly homologous subunits serve opposite functions. We demonstrated that only a few residues serve as "hotspots," initiating co-translational assembly interactions upon exposure at the ribosome exit tunnel. These hotspots are characterized by high binding energy, anchoring the entire interface assembly. Alpha-helices harbouring hotspots exhibited high levels of thermo-lability, folding and unfolding during simulations, depending on their partner subunit to avoid misfolding. In vivo hotspot mutations disrupted co-translational complex assembly. We next demonstrated that this assembly disruption led to subunits aggregation, analysed by advanced imaging approaches. Accordingly, conservation analysis revealed that missense NATs variants, causing neurodevelopmental and neurodegenerative diseases, disrupt putative hotspot clusters. Expanding our study to include more complexes, we find similar trends, allowing us to propose a predictive model.
To address aim (iii), we developed a single-molecule ribosome profiling approach, termed RiboFriend. We relied on our experience in combining biochemistry and sequencing to develop a nanopore based approach, to capture the interplay of factors guiding co-translational folding, in vivo. We have generated two chimeric constructs, fusing each co-translationally acting factor with a different RNA modifying enzyme. Direct RNA sequencing using NanoPore revealed that over 700 RNA transcripts exhibit significant mRNA modifications.
