Phase-separated ribosome-nascent chain complexes in genotoxic stress response

Abstract

Assemblysomes are EDTA- and RNase-resistant ribonucleoprotein (RNP) complexes of paused ribosomes with protruding nascent polypeptide chains. They have been described in yeast and human cells for the proteasome subunit Rpt1, and the disordered N-terminal part of the nascent chain was found to be indispensable for the accumulation of the Rpt1-RNP into assemblysomes. Motivated by this, to find other assemblysome-associated RNPs we used bioinformatics to rank subunits of Saccharomyces cerevisiae protein complexes according to their N-terminal disorder propensity. The results revealed that gene products involved in DNA repair are enriched among the top candidates. The Sgs1 DNA helicase was chosen for experimental validation. We found that indeed nascent chains of Sgs1 form EDTA-resistant RNP condensates, assemblysomes by definition. Moreover, upon exposure to UV, SGS1 mRNA shifted from assemblysomes to polysomes, suggesting that external stimuli are regulators of assemblysome dynamics. We extended our studies to human cell lines. The BLM helicase, ortholog of yeast Sgs1, was identified upon sequencing assemblysome-associated RNAs from the MCF7 human breast cancer cell line, and mRNAs encoding DNA repair proteins were overall enriched. Using the radiation-resistant A549 cell line, we observed by transmission electron microscopy that 1,6-hexanediol, an agent known to disrupt phase-separated condensates, depletes ring ribosome structures compatible with assemblysomes from the cytoplasm of cells and makes the cells more sensitive to X-ray treatment. Taken together these findings suggest that assemblysomes may be a component of the DNA damage response from yeast to human.

1,6-Hexanediol Is Inducing Homologous Recombination by Releasing BLM from Assemblysomes in Drosophila melanogaster

“…We selected the Sgs1 ortholog DmBLM as a candidate assemblysome component and quantified DmBLM mRNA using qPCR associated with assemblysomes and polysomes. The preparation of assemblysomes and polysomes involved ultracentrifugation of larval extracts through a sucrose cushion, following the procedure outlined in [1,2].…”

“…In brief, assemblysomes exhibit resistance to ethylenediaminetetraacetic acid (EDTA), enabling them to pass through a 60% sucrose cushion even in the presence of EDTA, which typically disconnects the small and large ribosome subunits, leading to mRNA accumulation at the top of the cushion [1,2]. All extracts were divided into two equal volumes and pelleted through sucrose cushions in the presence of EDTA (for assemblysomes) or in the absence of EDTA (for polysomes).…”

“…accumulation at the top of the cushion [1,2]. All extracts were divided into two equal volumes and pelleted through sucrose cushions in the presence of EDTA (for assemblysomes) or in the absence of EDTA (for polysomes).…”

“…We recently reported a significant discovery regarding the presence of DNA repair protein mRNAs in the cytoplasmic condensates of phase-separated ribosome nascent chain complexes (RNCs), referred to as assemblysomes [1,2]. It has been demonstrated that, in response to DNA damage, assemblysomes undergo a transformation into translating ribosomes that express DNA damage response proteins, such as the Sgs1 helicase [1]. Overexpression of BLM-which has functional similarities with DmBLM (in Drosophila melanogaster) and Sgs1 (in Saccharomyces cerevisiae)-is known to induce DNA damage [3][4][5][6][7][8][9].…”

“…The compound 1,6-hexanediol (HEX), a potent substance used to dissolve phaseseparated organelles like P-bodies and stress granules (SGs), was found to reduce ringoriented ribosomes reminiscent of assemblysomes in the A549 tumor cell line [1,13]. This reduction in parallel mitigated the DNA damage response in these otherwise radiationresistant cells [1]. HEX has been shown to disturb liquid-liquid phase separation (LLPS) in the Drosophila singe cell system [14].…”