The role of RNA-binding proteins in the nucleolar stress response

The central dogma of cell biology is the information in the genetic material in the cell, the DNA, is converted via an intermediary substrate, mRNA, into proteins. For proteins to be synthesised the mRNA must interact with a large complex called the ribosome which consists of ribosomal RNA (rRNA, which do not carry instructions to make specific proteins like mRNAs but instead provide structure and contribute to the activity of the ribosome) and proteins. Ribosomes can be thought of as molecular factories where the genetic information that is held in the mRNA is decoded and then synthesised into proteins. Ribosomes are essential for cell growth and proliferation and for a cell to divide and grow, it is necessary for the number of ribosomes to double (so that more proteins can be made). For this to occur it is essential that all the proteins that make up the ribosome (there are approximately 80 of these) are made at the same time, in the correct amount and the right amount of rRNA has to be synthesised. The rRNA is made in the nucleus within a compartment that is called the nucleolus. The protein that is used to read the sequences in the DNA and covert them into rRNA in called polymerase I (Pol I).

When cells are exposed to external agents that can damage them (such a sunlight or following viral infection), they undergo a complex process where they sense the damage that is caused and the proteins in the cells act to bring about a range changes to ensure the survival of the cell. During this time the cell stops growing and dividing and one of the ways in which this is achieved is by stopping the production of rRNA by inhibition Pol I activity and stopping the production of ribosomes. When this occurs the nucleolus is disassembled and the proteins within the nucleolus enter the rest of the nucleus (the nucleoplasm). While most of the proteins in the nucleolus are simply required to make ribosomes, some of these proteins have important other functions. Our data, and that of others, suggest that non-ribosomal functions of some of the proteins in the nucleolus are associated with the cellular responses to the damage that has been caused to it by external agents. In this application we propose to study how these proteins with non-ribosomal functions allow the cell to respond to different types of injury. The results obtained from these studies could have important implications for devising new ways in which to target disease that are associated with aberrant cell proliferation such as cancers.

The nucleolus is a subnuclear region whose major function is the production of ribosomes. Ribosome biogenesis consumes a large amount of cellular energy and this process is tightly linked to cell growth and proliferation. Ribosome biogenesis is very responsive to changes in environmental conditions and cell stress caused by many different types of agents (e.g. metabolic stress, genotoxic damage, viral infection) affects rRNA transcription making the nucleolus the central hub for coordination of the stress response. Inhibition of ribosome biogenesis results in a cascade of nucleolar led events that either maintain homeostasis or, if the degree of injury is too severe, initiate an apoptotic cascade. These molecular events are brought about by multitasking proteins, which shuttle from the nucleolus to the nucleoplasm, where they interact with their binding partners to orchestrate cell fate. However, the molecular mechanisms that link the disruption of rRNA production to cellular outcomes are not well defined. We have identified 170 RNA-binding proteins (RNA-BPs) within nucleoli that have additional functions (and do not appear to have direct roles in ribosome biogenesis). Many of these proteins have direct/indirect roles in the control of gene transcription. This has allowed us to suggest a model whereby the multitasking RNA-BPs within the nucleolus bind directly to rRNA under control conditions and are released into the nucleoplasm following cell stress where they control gene expression, and thus provide a direct link between ribosome biogenesis, cell stress/injury detection and cell outcome. In this proposal we will test this hypothesis by i) dissecting interactions of the multitasking nucleolar RNA-BPs with rRNA, ii) identifying their binding partners in the nucleoplasm and iii) determining what cell outcomes are controlled by these proteins; for example are they required for gene expression programmes that drive cell survival or cell death.

1. Academic beneficiaries: The main academic beneficiaries of this research would be those who work in gene regulation, systems biology and RNA biology.

2. Cancer Patients: Pol I inhibitors are being developed as anti-cancer agents. As the proteins to be studied are downstream of Pol I inhibition our research has the potential to impact on drug validation and discovery research programmes in academic and industrial settings. In the longer term, data generated from our research could impact on the quality of life and lifelong wellbeing and therefore lead to benefits to the wider public.

2. Biotechnology and pharmaceutical sectors: In the longer term our research could provide leads for new therapeutic targets and drug development programmes for cancer treatments.

3. Contribute to a highly trained workforce: The PDRA and RA will learn a wide range of state-of-the-art techniques and their skills will be invaluable for their future careers, should they pursue these in the pharmaceutical industry or academia. In addition, the transferable skills they learn will be relevant to a number of different settings, including a range of businesses.

4. The general public: It is essential to engage with the public so that they can understand and become fully conversant with the excellent science carried out in the UK, and so increase the knowledge economy. We will ensure that the public are fully informed with our research via lectures and social media such as Twitter and YouTube.

5. A high level of scientific knowledge and the new paradigms generated from this research will also benefit undergraduate and postgraduate teaching, both within the UK and worldwide.