Investigating how pertubations in ribosome biogenesis and the nucleolar surveillance response modulate cellular growth
Ribosome biogenesis is a key component in the synthesis of cellular proteins, and is an absolute requirement for cellular growth and proliferation. Perturbations in ribosome biogenesis, either by inhibiting Pol I transcription (involved in ribosomal RNA synthesis), or defective or absence of ribosomal proteins, leads to the activation of the ‘nucleolar surveillance pathway’, where MDM2 is sequestered by free ribosomal proteins, allowing p53 to become stabilised and transcription of p53 target genes that cause cell cycle arrest, apoptosis or senescence responses (Hein et al., Trends Mol Med 2013).
We have demonstrated that cancer cells are associated with dysregulated ribosome biogenesis, and that targeting Pol I transcription with a small molecule inhibitor (CX-5461) leads to p53-mediated apoptosis of cancer cells, whilst having no effect on normal cells (Bywater et al., Cancer Cell 2012). We have also demonstrated that knockdown of ribosomal proteins, such as RPS19 (mutated in Diamond-Blackfan anaemia) leads to p53-mediated cell cycle arrest. We believe that the mechanism underlying both of these observations is similar, i.e. due to nucleolar stress. However, much detail about this process is still lacking. As such, we have performed genome-wide short interfering RNA (siRNA) (loss of function) screens which have identified candidates that when absent (i) increase the activation of p53 and (ii) bypass the activation of p53 when ribosome biogenesis is disturbed with RPS19 knockdown.
This project will investigate the mechanism of action of a number of the candidates identified from the siRNA screen and how they regulate p53 expression. We will use techniques including but not limited to: cell culture, RNA interference, SDS- PAGE and western blotting, real-time qRT-PCR, FACS analysis and immunofluorescence /high-content imaging analysis to validate their role in blocking cancer cell growth, and overcoming perturbations in ribosome biogenesis.