Regulation of NF-kB signalling by ribotoxic stress

NF-kB转录因子家族的激活形成了生物体抵御环境威胁的第一道防线之一，并有助于编程适当的细胞反应。虽然最为人所知的是炎症反应的关键调节因子，但转录因子的NF-κ B家族也被细胞应激如缺氧、DNA损伤激活，并在衰老中发挥重要作用。NF-kB的异常激活与许多炎性疾病相关。5S RNP是核糖体的必需亚复合物，由5S核糖体（r）RNA和核糖体蛋白RPL 5和RPL 11组成。该复合物也具有重要的信号传导作用，控制信号传导因子如p53和cMyc，以响应核糖体产生的变化（称为核仁或核糖毒性应激）。核糖体的产生在许多形式的细胞应激反应中被阻断或受损。RPL 5和RPL 11的突变在诸如钻石黑扇贫血的疾病中被发现。然而，NF-kB和5S RNP之间的功能联系以前没有描述过。Perkins和Watkins小组之间的合作已经证明，p52 NF-kB亚基，替代NF-kB途径的一个组成部分，与5S RNP复合物相互作用，并且这种相互作用受到紫外线照射的刺激。来自Perkins实验室的初步数据也表明，5S RNP可以控制NF-κ B活性。因此，我们的数据定义了NF-κ B功能和核糖体产生之间的一个以前未知的调节联系，并表明5S RNP作为一个控制多个主要信号转导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。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）后更广泛的信号作用（罗查实验室，利物浦）。在纽卡斯尔（Perkins/Watkins labs），将进行诱变，以在体外和细胞中确定介导其相互作用的p52和RPL 5和/或RPL 11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新产生突变，从而特异性地破坏这种相互作用。在罗查实验室早期工作的基础上，这些细胞系将用于研究DNA损伤、缺氧和核糖毒性应激后p52/5S RNP复合物的重要性。这将包括使用RNA Seq分析基因表达和通过染色质免疫沉淀（ChIP）分析启动子占用。

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).