Regulation of NF-kB signalling by ribotoxic stress

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).