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

MRNAs的稳态水平受其合成和降解之间的平衡控制。MRNAs的正常衰变始于Poly(A)尾部的去烯化，随后是解帽和依赖Xrn1的5-3降解。这一途径确实发生在共翻译中(Pelachano等人，2015年细胞)。第二条途径涉及胞质外体及其辅助因子，即5-3降解的SKI复合体。(Schmidt等人，2016，科学)。核糖体-SKI复合体的结构表明，SKI复合体与小的核糖体亚单位结合，并在mRNA进入位点与出现的mRNA 3‘端结合。此外，我们还表明SKI复合体对携带小mRNA3悬垂的核糖体具有较好的亲和力。这些结果与SKI-核糖体复合体参与异常mRNAs的降解非常吻合，但考虑到SKI复合体在正常5-3mRNA衰退中的已知作用，这些结果令人惊讶。事实上，在这一途径中，在死烯基化之后，SKI-exosome复合体在其3-UTR处开始降解mRNAs，基本上没有核糖体。在这个阶段，Ska1将离开，并允许SKI复合体和核糖体之间的直接相互作用。当靶序列被核糖体占据时，这种构型显然是SKI-Exosome复合体活性所必需的。在这里，我们提出了一个新的合作项目，旨在通过遗传和全基因组筛选、亲和纯化、质谱学和冷冻电子显微镜来表征和比较核糖体相关的SKI复合体和Ska1相关的SKI复合体的生化、结构和功能。

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.