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

mrna的稳态水平是由其合成和降解之间的平衡控制的。mrna的正常衰变始于聚(A)尾部的死基化，随后是脱冠和依赖xrn1的5-3降解。这一途径被证明是共翻译的（Pelachano et al., 2015 Cell）。第二种途径涉及细胞质外泌体及其辅助因子，用于5-3降解的SKI复合物。SKI复合体也是异常mRNA翻译依赖性降解的关键参与者，例如缺乏停止密码子的mRNA （STOP mRNA Decay， NSD）或核糖体停止的mRNA （No-Go Decay， NGD）。我们（合作伙伴1和2）与Elena Conti小组合作，通过生物化学和高分辨率冷冻电镜显示了SKI复合物和核糖体之间的直接物理相互作用。（Schmidt et al., 2016, Science）。核糖体-SKI复合物的结构表明，SKI复合物在mRNA进入位点与小核糖体亚基和mRNA的新兴3'端结合。此外，我们发现SKI复合物对携带小mRNA 3悬垂的核糖体具有首选亲和力。这些结果与SKI-核糖体复合物参与异常mRNA的降解非常吻合，但考虑到SKI复合物在正常5-3 mRNA降解中的已知作用，这些结果令人惊讶。事实上，在这一途径中，在死基化之后，已知ski -外显体复合物开始降解mrna的3-UTR，这基本上是缺乏核糖体的。这个提议的目的之一就是解决这个明显的悖论。最近，我们发现Ska1 （SKI相关因子1）与独立于核糖体的SKI复合物的一个子集相关，这表明SKI复合物至少存在于两种不同的生化环境中，一种与核糖体相关，另一种与Ska1相关。这些观察结果与功能初步数据一致，显示Ska1是有效降解报告mRNA的3-UTR所必需的，而Ska1的缺失对NSD没有影响，NSD是一个依赖于翻译和SKI复合物的途径。我们提出了一个工作假设，根据该假设，在mRNA死基化后，Ska1-SKI复合物将帮助外胞体降解无核糖体的3-UTR mRNA区域，直到它到达编码区，在那里它会遇到核糖体。在这个阶段，Ska1会离开并允许SKI复合体和核糖体之间的直接相互作用。当目标mRNA序列被核糖体占据时，这种结构显然是ski -外泌体复合物活性所必需的。在此，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、质谱和低温电镜技术，从生物化学、结构和功能上表征和比较核糖体相关和ska1相关的SKI复合物。

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.