Gyorkei, A. | bioRxiv Science

bioRxiv2022

Phase separated ribosome nascent chain complexes paused in translation are capable to continue expression of proteins playing role in genotoxic stress response upon DNA damage

EDTA- and RNase-resistant ribonucleoprotein complexes of arrested ribosomes with protruding nascent polypeptide chains have recently been described in yeast and human cells. These complexes have been termed assemblysomes, a type of soluble condensates distinct from other known granules. Here, we use bioinformatics to identify additional proteins that likely form assemblysomes during translation. We characterize soluble condensates of the DNA helicase Sgs1, one such identified protein and a key player in the repair of DNA double-strand breaks in yeast. We show that paused ribosome-associated nascent chains of Sgs1 in condensates are able to resume translation upon UV irradiation, consistent with the return of mRNA to the ribosome pool. By extending our studies to human cell lines, we found that EDTA-resistant pellets of ribosomes from the human prostate cancer cell line DU145 are sensitive to treatment with 1,6-hexanediol, which is known to dissolve liquid-liquid phase-separated condensates. In addition, transmission electron microscopy shows that 1,6-hexanediol dissolves ring ribosomal structures from the cytoplasm of radioresistant A549 cells while making the cells more sensitive to X-rays. These results suggest that the stress response is based on a conserved mechanism involving the regulated return of phase-separated paused ribosome-nascent chain complexes to translating ribosomes.

molecular biology↗

bioRxiv2021

Proteome-wide landscape of solubility limits in a bacterial cell

Proteins are prone to aggregate when they are expressed above their solubility limits, a phenomenon termed supersaturation. Aggregation may occur as proteins emerge from the ribosome or after they fold and accumulate in the cell, but the relative importance of these two routes remain poorly known. Here, we systematically probed the solubility limits of each Escherichia coli protein upon overexpression using an image-based screen coupled with machine learning. The analysis suggests that competition between folding and aggregation from the unfolded state governs the two aggregation routes. Remarkably, the majority (70%) of insoluble proteins have low supersaturation risks in their unfolded states and rather aggregate after folding. Furthermore, a substantial fraction ([~]35%) of the proteome remain soluble at concentrations much higher than those found naturally, indicating a large margin of safety to tolerate gene expression changes. We show that high disorder content and low surface stickiness are major determinants of high solubility and are favored in abundant bacterial proteins. Overall, our proteome-wide study provides empirical insights into the molecular determinants of protein aggregation routes in a bacterial cell.

molecular biology↗