Structural and functional characterization of a SKI sub complex in Saccharomyces cerevisiae

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The steady state level of mRNAs is controlled by the equilibrium between its synthesis and degradation. The normal decay of mRNAs starts with deadenylation of the poly(A) tail, followed by decapping and Xrn1-dependent 5-3 degradation. This pathway and was shown do occur co-translationally (Pelachano et al., 2015 Cell). A second pathway involves the cytoplasmic exosome and its co-factor, the SKI complex for 5-3 degradation. The SKI complex is also a key player of translation-dependent degradation of aberrant mRNAs, such as mRNAs lacking a STOP codon (Non-Stop mRNA Decay, NSD) or mRNAs on which ribosomes are stalled (No-Go Decay, NGD).We (Partners 1 & 2), in collaboration with the group of Elena Conti, have shown the direct physical interaction between the SKI complex and the ribosome biochemically and by high resolution cryo-EM. (Schmidt et al., 2016, Science). The structure of the ribosome-SKI complex shows, that the SKI complex binds to the small ribosomal subunit and to the emerging 3'-end of mRNA at the mRNA entry site. Moreover, we show the preferred affinity of the SKI complex for ribosomes carrying small mRNA 3 overhangs. These results fit very well with the involvement of the SKI-ribosome complex in the degradation of aberrant mRNAs, yet they are surprising given the known role of the SKI complex in normal 5-3 mRNA decay. In fact, in this pathway and following deadenylation, the SKI-exosome complex is known to start to degrade the mRNAs at their 3-UTR, which is essentially devoid of ribosomes. One of the aims of this proposal is to address this apparent paradox.Very recently, we identified Ska1 (SKI associated factor 1) to be associated with a subset of the SKI complexes independently of the ribosome, suggesting that the SKI complex could be found in at least two distinct biochemical environments, one in association with the ribosome, one in association with Ska1. These observations are consistent with functional preliminary data showing that Ska1 is required for the efficient degradation of the 3-UTR of a reporter mRNA, while its absence has no effect on NSD, a pathway dependent on both translation and the SKI complex.We propose a working hypothesis according to which, after mRNA deadenylation, the Ska1-SKI complex would assist the exosome to degrade the ribosome-free 3-UTR mRNA regions, until it reaches the coding regions where it would encounter ribosomes. At this stage, Ska1 would leave and allow the direct interaction between the SKI complex and the ribosome. This configuration is apparently required for the activity of SKI-exosome complexes when the targets mRNA sequences are occupied by ribosomes.Here we propose a novel collaborative project with the aim to characterize and compare the ribosome-associated and the Ska1-associated SKI complexes biochemically, structurally and functionally by applying genetic and genome-wide screens, affinity purification, mass spectrometry and cryo-electron microscopy.