Exosome-Ski-Complex · Structural and functional studies on how the Ski complex activates the exosome to degrade RNA
FP7 — People (Marie Curie Actions)
- Duration
- 2013-07-01 → 2015-06-30
- EU contribution
- €161,969
- Participants
- 1
- Scheme
- MC-IEF
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Results in brief
Structural and functional studies on how the Ski complex activates the exosome to degrade RNA
Objectives of the completed project The aim of the completed project was to understand how the 10 subunit exosome (Exo10) and Ski complex function together to degrade RNA substrates. The main objectives are to find out: • How are the catalytic activities (a nuclease and a helicase) organized and how do they communicate? • How is an RNA substrate recruited, unwound and degraded? • How does the Ski complex provoke RNA degradation? • What is the role of Ski7? A combination of structural and functional studies of the Ski2387Exo10 complex has been employed to answer these questions. We expected to acquire a crystal- or electron microscopy structures of the entire ca. 800 kDa complex or sub-complexes of the Ski2387 and Ski7Exo10 as a basis for functional in vitro and in vivo studies that should elucidate the functioning of this important cellular RNA degradation machinery. Outcome of the project In the course of the project, we were able to solve several crystal structures and gain structural information on the whole molecule of Ski7, from N to C-terminus. The structures lead especially to a better understanding of the role of Ski7 in the cell but moreover help to model more precisely the spacial arrangement of the Ski-complex and exosome. • With its very N-terminal domain, Ski7 binds the Ski-complex, exclusively interacting with the C-terminal portion of Ski3. • With the second part of the N-terminus, the cytosolic Ski7 wraps around the exosome in a manner very similar to its nuclear counterpart Rrp6 although their sequences are very different. • The C-terminal GTP-binding domain of Ski7 is not involved in the interaction of Ski-complex and exosome. It carries the fold of a typical translational GTPase, like its closest homologue Hbs1. However in the active site of Ski7, residues crucial in GTP hydrolysis are not conserved and Ski7 might have evolved for stable GTP binding (in collaboration with the laboratory of Rachel Green, Baltimore, USA) and binding to the ribosome. In conclusion the Ski7 N-terminus seems to act solely as adaptor between both complexes not interfering with their catalytically active moieties. Combining the two the substructures lead to an improved working model for the holo-complex. Furthermore, we found that the Ski-complex can directly bind to the ribosome, having a co-translational function, probably when ribosomes get stalled on faulty messenger RNA (mRNA) and a cryo electron microscopic structure of the Ski-complex on the ribosome was solved (in collaboration with the laboratory of Roland Beckmann, LMU Munich, Germany).
Data: CORDIS, © European Union
Project objective
Regulation of RNA stability is recognized as a widely used strategy to regulate protein abundance in time and space in all eukaryotes. RNA degradation regulates gene expression in various processes during development and differentiation and in changing physiological conditions. Many elements of the RNA decay machinery have been identified, but details of their function remain elusive.The exosome is a multi-subunit exonuclease complex that functions in turnover, quality control and processing of many RNA species. Depending on its subcellular localization it requires different accessory protein complexes that specifically target RNAs towards the exosome and stimulate the degradation process. In the yeast cytosol the Ski complex is the major accessory factor of the exosome. It is required for nearly all exosome-mediated mRNA degradation processes. The core Ski complex is formed by Ski2, an RNA helicase, the large TPR protein Ski3 and two copies of Ski8. This core complex interacts genetically and physically with the exosome via the GTPase Ski7.In the proposed project, I will study how the activities of the Ski238 complex, Ski7p and the exosome are concerted. For this I will recombinantly express, purify and crystallize the Ski2387 complex alone and together with the exosome to learn about their interaction on atomic level. This approach structural approach will be complemented by electron microscopic studies of the complexes. The results of the structural analysis will guide the design of subsequent functional biochemical and in vivo studies.The study will be exemplary for the understanding of the functioning of an important cellular machinery in atomic detail.
Original text from CORDIS.
Participants
- MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV · MUNCHENCoordinatorGermany
Links
Data: CORDIS, © European Union