Mechanistic investigation of eukaryotic quality control factors RQT and Mbf1

Best, Katharina (2023): Mechanistic investigation of eukaryotic quality control factors RQT and Mbf1. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

Translation is a central cellular process and thus tightly regulated by quality control mechanisms. Monitoring the ribosome during translation is an elegant way to track the progress and to catch a variety of errors before they lead to detrimental effects in the cell. Recently, ribosomal collisions have emerged as a trigger for such quality control pathways, and various collision sensors have been identified. One of those pathways called ribosome associated quality control (RQC) deals with ribosomes stuck on an open reading frame (e.g. due to stable mRNA structures or inhibitory codons). Here, ubiquitination of ribosomal proteins serves as a signal for dissociation of the stuck ribosome by the RQC-trigger (RQT) complex. Subsequently the aberrant mRNA and the truncated nascent peptide are degraded and intact components such as ribosomal subunits or tRNAs can be recycled. Although this pathway has been studied in detail over the last years, the exact mechanism by which RQT leads to dissociation of stalled ribosomes remains unclear. This thesis aimed to elucidate the RQT-mediated dissociation mechanism by setting up an in vitro splitting system and subsequent cryo-EM analysis of the splitting reactions. To generate suitable substrates for the dissociation process, collisions were generated using known ribosome stalling sequences in a cell free in vitro translation system. Splitting assays showed that an in vitro ubiquitination step for collided ribosomes is crucial for splitting. Moreover, such assays revealed that efficient splitting is dependent on ATPase activity of the N-terminal helicase cassette of RQT component Slh1, on the presence of a neighboring ribosome and on availability of a 3’ mRNA overhang. Structural analysis of the ribosome-bound RQT complex divulged stable positioning of RQT on the lead ribosome of a collided ‘disome’ unit, as well as on 80S and 40S. The 80S-RQT complex was observed in two different states located in close proximity to the entry of the mRNA channel. Together with the observed requirement of available 3’mRNA and helicase activity of Slh1, this suggests that Slh1 can pull on the mRNA, leading to an initial model for ribosome dissociation. Ribosome stalling and subsequent collisions increase the probability of frameshifting and thus translation of an aberrant protein. Structural analysis of three collided ribosomes, so called trisomes, revealed the presence of multiprotein bridging factor (Mbf1), previously identified as a frameshift inhibitor. This small protein was found on the second and third colliding ribosomes, positioned between beak and body of the 40S subunit. Comparison with the human homolog EDF1, which was found to be recruited to emetine induced collisions, showed that those proteins bind in the exact same fashion. The position on the 40S subunit of the collided ribosomes suggests that both proteins interact directly with the mRNA to prevent frameshifting, probably in combination with preventing conformational changes required for translocation of the ribosome.   In conclusion, high resolution cryo-EM structures of both RQT and Mbf1 on ribosomes enabled detailed insights into the intricate quality control network targeting collisions in the cell. From this, molecular models for both a helicase driven dissociation mechanism by RQT and the frameshifting inhibition by Mbf1 could be derived. These results, together with the developed optimization strategies, provide the basis for future works, leading to a detailed understanding of these pathways.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 27. April 2023
• 1. Bericht­erstatter:in: Beckmann, Roland