Translation inhibition during ribotoxic stress mediated by the CAF-1 complex

• Applicant: Dr. Martin Müller
• Subject Area: Biochemistry ; Structural Biology ; Cell Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 549429015

Translation comprises three phases – initiation, elongation and termination. Each translation cycle starts with the formation of 43S initiation complexes, which scan for start codons to initiate nascent chain elongation. At the end of each translation cycle, translation terminates when ribosomes reach stop codons, leading to the release of nascent chains and subsequent ribosome splitting. Translational fidelity decreases with ageing and the termination process becomes less efficient, leading to translation past stop codons. Resulting protein products may be nonfunctional, as C-terminal extensions can interfere with their native fold. Cells have evolved a defensive mechanism to detect and resolve such translation events. I recently discovered a novel mechanism that clears potentially corrupted mRNAs that undergo stop codon readthrough and simultaneously degrades the resulting protein products. However, it remains unclear whether a cis-acting mechanism exists, blocking translation initiation to prevent the production of further corrupted protein species. I have compelling evidence that such a cis-acting mechanism indeed exists, mediated by the CAF-1 complex. This complex resides normally in the nucleus, but shuttles into the cytosol during stress conditions like UV treatment. Yet, what CAF-1 does in the cytosol remains unexplored. I found that during chemically enhanced readthrough, this complex appears to be recruited to translating ribosomes. Reanalysis of an unbiased genetic screen for stop codon readthrough quality control components identified the CAF-1 complex as a potential candidate. Furthermore, orthogonally tethering components of CAF-1 to mRNAs blocks translation. Interactome analysis of a CAF-1 component identified the eukaryotic initiation factors EIF4G1 and EIF4A1 as strong interactors, supporting the idea of CAF-1 acting as a translation initiation modulator. Taken together, these data suggest that CAF-1 may inhibit translation in response to stop codon readthrough. In this proposal, I aim to further explore the mechanism of how the CAF-1 complex executes stop codon readthrough quality control. I plan to characterize the context in which CAF-1 inhibits translation inhibition, testing different stressors that may activate this mechanism. For CAF-1 to function in the cytosol, it first needs to translocate from the nucleus. While it has been proposed that phosphorylation precedes CAF-1 translocation into the cytosol, the exact mechanism remains unclear. Using hypothesis-driven and unbiased genetic approaches, I aim to identify the underlying mechanism of CAF-1 translocation. To answer the question how CAF-1 acts on the ribosome, I aim to solve the structure of CAF-1 on ribosomes, or in complex with EIF4G1 and EIF4A1. In the last step, using selective riboseq I aim to identify substrates of CAF-1 mediated translation initiation inhibition. Using these approaches, I will characterize CAF-1 as a translational repressor during stress.

• DFG Programme: WBP Position