Regulation of NF-kB signalling by ribotoxic stress

NF-kB转录因子家族的激活是机体抵御环境威胁的第一道防线之一，并有助于规划适当的细胞反应。尽管NF-kB家族转录因子被认为是炎症反应的关键调节因子，但它也会被细胞应激（如缺氧、DNA损伤）激活，并在衰老过程中发挥重要作用。NF-kB的异常活化与许多炎症性疾病有关。5S RNP是核糖体的重要亚复合物，由5S核糖体(r)RNA和核糖体蛋白RPL5和RPL11组成。该复合体还具有重要的信号作用，控制诸如p53和cMyc等信号因子，以响应核糖体产生的变化（称为核核或核毒应激）。在多种形式的细胞应激反应中，核糖体的产生被阻断或受损。RPL5和RPL11的突变在一些疾病中被发现，比如钻石黑扇贫血。然而，NF-kB和5S RNP之间的功能联系以前没有被描述过。Perkins和Watkins小组之间的合作已经证明，p52 NF-KB亚基是NF-KB替代途径的一个组成部分，与5S RNP复合物相互作用，这种相互作用受到紫外线照射的刺激。珀金斯实验室的初步数据也表明，5S RNP可以控制NF-KB的活性。因此，我们的数据定义了NF-KB功能和核糖体产生之间先前未知的调控联系，并表明5S RNP作为控制多种主要信号通路的中心枢纽发挥作用。在这个项目中，学生将研究p52和5S RNP复合物的性质和功能。该学生将首先研究5S RNP在生理细胞应激（如缺氧和炎症细胞因子刺激）下更广泛的信号作用（利物浦Rocha实验室）。在纽卡斯尔（Perkins/Watkins实验室），将在体外和细胞中进行诱变，以确定p52和RPL5和/或RPL11介导其相互作用的结构域。然后将进行CRISPR/Cas9基因组工程，以在内源性蛋白质中重建特异性破坏这种相互作用的突变。在Rocha实验室早期工作的基础上，这些细胞系将被用于研究p52/5S RNP复合物在DNA损伤、缺氧和核毒性应激后的重要性。这将包括使用RNA Seq和染色质免疫沉淀（ChIP）启动子占用分析基因表达。

Activation of the NF-kB transcription factor family forms one of the first lines of defence against environmental threats to the organism and helps programme an appropriate cellular response. Although best known as critical regulators of the inflammatory response, the NF-kB family of transcription factors are also activated by cellular stresses such as hypoxia, DNA damage and play an important role in ageing. Aberrant activation of NF-kB is associated with many inflammatory diseases. The 5S RNP is an essential subcomplex of the ribosome and is comprised of the 5S ribosomal (r)RNA and the ribosomal proteins RPL5 and RPL11. This complex also has an essential signalling role, controlling signalling factors such as p53 and cMyc, in response to changes in ribosome production (termed nucleolar or ribotoxic stress). Ribosome production is blocked or impaired in response to many forms of cellular stress. Mutations in RPL5 and RPL11 are found in diseases such as Diamond Blackfan anaemia. However, a functional link between NF-kB and the 5S RNP has not been previously described.A collaboration between the Perkins and Watkins groups has demonstrated that the p52 NF-KB subunit, a component of the alternative NF-kB pathway, interacts with the 5S RNP complex and that this interaction is stimulated by UV irradiation. Preliminary data from the Perkins lab has also suggests that the 5S RNP can control NF-KB activity. Our data therefore defines a previously unknown regulatory link between NF-KB function and ribosome production and indicates that the 5S RNP functions as a central hub that controls multiple major signalling pathways.In this project the student will investigate the nature and function of the p52 and 5S RNP complex. The student will first investigate the wider signalling role of the 5S RNP following physiological cellular stresses such as hypoxia and stimulation with inflammatory cytokines (Rocha lab, Liverpool). In Newcastle (Perkins/Watkins labs), mutagenesis will be performed to determine, in vitro and in cells, the domains of p52 and RPL5 and/or RPL11 that mediate their interaction. CRISPR/Cas9 genome engineering will then be performed, to recreate mutations in the endogenous proteins that specifically disrupt this interaction. Building on the earlier work in the Rocha lab, these cell lines will then be used to investigate the importance of the p52/5S RNP complex following DNA damage, hypoxia and ribotoxic stress. This will include analysis of gene expression using RNA Seq and promoter occupancy by chromatin immunoprecipitation (ChIP).