In this mini-review, we summarize our current knowledge about the cross-talk between the different levels of gene expression. We introduce the Ccr4 (carbon catabolite repressed 4)–Not (negative on TATA-less) complex as a candidate to be a master regulator that orchestrates between the different levels of gene expression. An integrated view of the findings about the Ccr4–Not complex suggests that it is involved in gene expression co-ordination. Since the discovery of the Not proteins in a selection for transcription regulators in yeast [Collart and Struhl (1994) Genes Dev. 8, 525–537], the Ccr4–Not complex has been connected to every step of the mRNA lifecycle. Moreover, it has been found to be relevant for appropriate protein folding and quaternary protein structure by being involved in co-translational protein complex assembly.

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 amino-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 ofSaccharomyces cerevisiaeprotein complexes according to their amino-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,SGS1mRNA 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.

We aimed to investigate the contribution of co-translational protein aggregation to the chemotherapy resistance of tumor cells. Increased co-translational protein aggregation reflects altered translation regulation that may have the potential to buffer transcription under genotoxic stress. As an indicator for such an event, we followed the cytoplasmic aggregation of RPB1, the aggregation-prone largest subunit of RNA polymerase II, in biopsy samples taken from patients with invasive carcinoma of no special type. RPB1 frequently aggregates co-translationally in the absence of proper HSP90 chaperone function or in ribosome mutant cells as revealed formerly in yeast. We found that cytoplasmic foci of RPB1 occur in larger sizes in tumors that showed no regression after therapy. Based on these results, we propose that monitoring the cytoplasmic aggregation of RPB1 may be suitable for determining—from biopsy samples taken before treatment—the effectiveness of neoadjuvant chemotherapy.

We recently demonstrated that 1,6-hexanediol inhibits the formation of assemblysomes. These membraneless cell organelles have important roles in co-translational protein complex assembly and also store halfway translated DNA damage response proteins for a timely stress response. Recognizing the therapeutic potential of 1,6-hexanediol in dismantling assemblysomes likely to be involved in chemo- or radiotherapy resistance of tumor cells, we initiated an investigation into the properties of 1,6-hexanediol. Our particular interest was to determine if this compound induces DNA double-strand breaks by releasing the BLM helicase. Its yeast ortholog Sgs1 was confirmed to be a component of assemblysomes. The BLM helicase induces DNA damage when overexpressed due to the DNA double-strand breaks it generates during its normal function to repair DNA damage sites. It is evident that storing Sgs1 helicase in assemblysomes is crucial to express the full-length functional protein only in the event of DNA damage. Alternatively, if we dissolve assemblysomes using 1,6-hexanediol, ribosome-nascent chain complexes might become targets of ribosome quality control. We explored these possibilities and found, through the Drosophila wing-spot test assay, that 1,6-hexanediol induces DNA double-strand breaks. Lethality connected to recombination events following 1,6-hexanediol treatment can be mitigated by inducing DNA double-strand breaks with X-ray. Additionally, we confirmed that SMC5 recruits DmBLM to DNA damage sites, as knocking it down abolishes the rescue effect of DNA double-strand breaks on 1,6-hexanediol-induced lethality in Drosophila melanogaster.

Assemblysomes are recently discovered intracellular RNA–protein complexes that play important roles in cellular stress response, regulation of gene expression, and also in co‐translational protein assembly. In this review, a wide spectrum overview of assemblysomes is provided, including their discovery, mechanism of action, characteristics, and potential applications in several fields. Assemblysomes are distinct liquid–liquid phase‐separated condensates; they have certain unique properties differentiating them from other cellular granules. They are composed of ribosome‐nascent protein chain complexes and are resistant to cycloheximide and EDTA. The discovery and observation of intracellular condensates, like assemblysomes, have further expanded our knowledge of cellular stress response mechanisms, particularly in DNA repair processes and defense against proteotoxicity. Ribosome profiling experiments and next‐generation sequencing of cDNA libraries extracted from EDTA‐resistant pellets—of ultracentrifuged cell lysates—have shed light on the composition and dynamics of assemblysomes, revealing their role as repositories for pre‐made stress‐responsive ribosome‐nascent chain complexes. This review gives an exploration of assemblysomes' potential clinical applications from multiple aspects, including their usefulness as diagnostic biomarkers for chemotherapy resistance and their implications in cancer therapy. In addition, in this overview, we raise some theoretical ideas of industrial and agricultural applications connected to these membraneless organelles. However, we see several challenges. On one hand, we need to understand the complexity of assemblysomes' multiple functions and regulations; on the other hand, it is essential to bridge the gap between fundamental research and practical applications. Overall, assemblysome research can be perceived as a promising upcomer in the improvement of biomedical settings as well as those connected to agricultural and industrial aspects.

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.