Structural and biochemical characterization of the human SKI complex

Kögel, Alexander (2022): Structural and biochemical characterization of the human SKI complex. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The eukaryotic RNA-degrading exosome is universally present in nuclear and cytoplasmic cellular compartments and is involved in wide-spread RNA processing and degradation functions that rely on its 3'-5' RNA exonuclease activity. In the cytoplasm, it associates with the Ski complex via the Ski7 protein to function in mRNA turnover and surveillance pathways. As part of the Ski complex, together with the Ski3 and Ski8 proteins, the DExH helicase Ski2 binds and threads RNA to the exosome for degradation. The collaboration of the respective helicase and nuclease activities is conserved in eukaryotes and has been well studied in S. cerevisiae, but major questions regarding the molecular mechanisms of its regulation and unwinding activity remain open. In this dissertation, we solved several cryo-EM structures of the human SKI (hSKI) complex in a resting state in absence of a substrate, in a substrate-binding but inactive state and in an active substrate-working state and uncovered the existence of two fundamentally different activity-related conformations. The open and closed conformations, are characterized by the detachment of the hSKI2 helicase core from the complex. In the closed state, the hSKI3 protein blocks the RNA exit site of the hSKI2 helicase like a gate to prevent threading of the substrate. In the open conformation, when the hSKI2 helicase is detached from the complex, the RNA exit site is free, allowing substrate translocation. When analyzed bound to the 80S ribosome, the inactive hSKI complex recognizes short RNA 3' overhangs at the mRNA entry site in the closed conformation. The activation of the complex the detaches the hSKI2 helicase and efficiently extracts RNA from the mRNA entry site of the ribosome. The crystal structure of the S. cerevisiae Ski7-bound cytoplasmic exosome shows the interaction of Ski7 with a conserved binding interface with the nuclear exosome adaptor Rrp6. Knowing the precise mode of interaction between Ski7 and the exosome, we identified a human Ski7-like protein in a splicing variant of the HBS1L protein that bridges the interaction between the hSKI complex and the human cytoplasmic exosome. In subsequent biochemical experiments, we further verified its function and study the RNA channeling capabilities of the hSKI-exosome holocomplex. We conclude that RNA channeling to the cytoplasmic exosome requires the open conformation of hSKI and that RNA channeling between the two complexes follows a conserved principle similar to the nuclear Mtr4-exosome holocomplex. In addition, the analysis and comparison to the homologous S. cerevisiae Ski complex allowed us to identify common principles of regulation with respect to the closed and open conformational states but also spots differences with respect to the auto-inhibition previously described in the S. cerevisiae complex. The findings of this dissertation give unique insight into the molecular mechanisms of the human SKI complex and change the current understanding of how it functions together with the exosome to degrade mRNAs in the cytoplasm. These insights furthermore set the basis to begin to understand how hSKI-related mutations give rise to human disease.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: SKI complex, Exosome, Ribosome, mRNA degradation, Cryo-EM
• 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. Mai 2022
• 1. Bericht­erstatter:in: Conti, Elena