Alternative 18S rRNA 3' processing pathways in human ribosome biogenesis

Life depends on the ability of an organism to react to changing environments and stresses by controlling the synthesis of complex macromolecules. Normal cell growth requires the production of ribosomes, the essential RNA-protein machinery that makes all cellular proteins. Ribosome assembly, a complicated process involving hundreds of biogenesis factors, is the major consumer of cellular energy and therefore tightly controlled. In cancer cells, ribosome production is upregulated to promote cell proliferation. Many cellular stresses and 20 genetic diseases ("ribosomopathies") result in a block or defect in ribosome production. Surprisingly, ribosomopathy patients are often pre-disposed to cancer and some cancers have also recently been linked to defective ribosome production. In humans, ribosome assembly controls the tumour suppressor p53, the "guardian of the genome". Through cellular signaling, p53 ensures appropriate cellular responses to stress conditions, and can even induce cell death if individual cells are detrimental to the whole organism. Indeed, p53 levels are elevated in many ribosomopathies and p53 is essential for many of the clinical features observed in these diseases.

Here, we propose to study the impact of disease-associated mutations in key biogenesis factors on human ribosome production, p53 and cellular survival. Our internationally recognised expertise in ribosome biogenesis, RNA processing and cellular signaling, together with our strong preliminary data, mean that we are ideally placed to successfully carry out this project.

Due to the high degree of evolutionary conservation in the ribosome production machinery, it was long assumed that ribosome production is the same in humans as it is in yeast, the main model organism used to study this process. However, we have now shown that the process of human ribosome production is significantly more complicated, and fundamentally different to that used in yeast. Surprisingly, human cells also retain the machinery needed for a "yeast-like" mechanism, however, this pathway is rarely used in humans and it is unclear what role it plays. Increasing our fundamental knowledge of human ribosome production is therefore of paramount importance for elucidating the basis of several human diseases and the development of potential treatments.

WE WILL THEREFORE ADDRESS TWO KEY QUESTIONS:
1) Do disease-associated mutations in key ribosomal RNA maturation factors cause defects or changes in ribosome production?
2) Why do human cells use a specialised ribosomal RNA maturation pathway when they also have the machinery to employ a "yeast-like" mechanism?

We will use a powerful combination of molecular tools and state-of-the-art technologies to reveal the mechanism(s) of human ribosome production and understand how this is altered in disease. This project will include determining the impact of disease-associated mutations on human ribosome production and p53-dependent cellular signaling, as well as using novel approaches to understand why human cells use a specialised ribosome production pathway.

Intriguingly, our preliminary data also suggest that some cancer cells might depend on the minor, "yeast-like" mechanism. This raises the exciting possibility that this pathway could be targeted for the development of anti-cancer drugs in the future.

Ribosomes, the machines responsible for protein synthesis, are essential for cell growth and function. Ribosome maturation is the most energetically expensive process in the cell and controls many cellular signalling pathways linked to proliferation, such as p53 and c-Myc. Budding yeast has been the paradigm for ribosome biogenesis for decades and it was assumed that the human ribosome production pathway is the same as in yeast as many of the factors involved in pre-ribosomal RNA (pre-rRNA) processing are conserved throughout eukaryotes. However, we have shown that the predominant mechanism for 18S rRNA 3' end processing is specific to higher eukaryotes and distinct from that found in yeast. In human cells, there is a major 18S 3' rRNA processing pathway and a minor, "yeast-like" pathway. This finding is highly relevant as 18S rRNA 3' processing is impaired in many of the 20 genetic diseases (ribosomopathies) resulting from mutations in genes encoding ribosomal proteins and ribosome biogenesis factors. A common feature of ribosomopathies is that the patients are pre-disposed to cancer. Indeed, mutations similar to those found in ribosomopathy patients have been found in a wide range of tumours.

Our preliminary data suggest that disease-associated mutations may either impair 18S 3' rRNA maturation or switch the predominant, major pathway to a minor, "yeast-like" mechanism. Revealing the dependence of cancer cells on the minor pathway would offer the potential of targeting factors involved in this process to develop anti-cancer drugs. This exciting and timely project will employ a powerful combination of mutagenesis, proteomics and high-throughput protein-RNA interaction studies as well as a novel RNA modification interference method to specifically block individual pre-rRNA processing events. Our findings on the mechanisms in human 18S rRNA 3' processing will contribute significantly to the understanding of how defects in this process contribute to human disease.

To achieve the maximum impact for the work outlined in this proposal we will:
1) Make sure that our findings are disseminated effectively across the academic community.
2) Ensure that we protect and exploit commercially valuable intellectual property derived from our work relevant to human disease.
3) Actively contribute to public outreach programmes to enhance public understanding of the link between ribosome production and human disease.
4) Build on our collaborations both in the UK and abroad.
5) Provide both excellent training and a superb environment for the PDRA to prosper in the area of ribosome production, cellular signalling and human disease.

1) ACADEMIC DISSEMINATION: We will make sure that our work is disseminated as broadly as possible across the academic community by publishing in leading, open access journals, presenting at key national and international conferences in the relevant fields and collaborating with colleagues both nationally and internationally.
2) RELEVANCE TO HUMAN HEALTH AND COMMERCIAL EXPLOITATION: The project outlined here addresses an important area of human health. We aim to understand how mutations in the genes encoding key factors essential to ribosome production lead to human disease. The research in this proposal is at an early, 'discovery' stage, and focused on understanding the basic science underpinning the links between ribosome production, cellular signalling and disease.
Defects in ribosome biogenesis have been linked to 20 genetic diseases ("ribosomopathies") including Diamond Blackfan anaemia, Treacher Collins syndrome and 5q syndrome. These diseases show a wide variety of clinical symptoms, but the most frequent traits are anaemia, skeletal defects and a predisposition to cancer, likely due to mis-regulation of the tumour suppressor p53. Indeed, mutations in ribosomal proteins or ribosome biogenesis factors are found in many cancers. Most noteworthy is nucleophosmin (NPM1), which is mutated in one third of all leukaemias. It is unclear how defects in ribosome biogenesis lead to specific clinical symptoms seen in ribosomopathies. Furthermore, ribosome production is upregulated in cancer. It is therefore somewhat of a paradox that mutations that would impact this process and result in p53 activation, would predispose the patients to cancer. The project directly addresses these points and aims to provide a better understanding about the impact of ribosome production defects, caused by mutations in the genes of key ribosome biogenesis factors, on cellular growth, cell signalling and cancer. This is important in light of the recently published high-throughput sequencing study used to analyse the genomes of whole cohorts of cancer patients.
3) PUBLIC OUTREACH: Increasing the public understanding and awareness of scientific research is something that we strongly believe in. This will continue with this project where we will contribute on both a local and national level as described in detail in the Pathways to Impact statement.
4) COLLABORATIONS: We have a wide variety of both national and international collaborators. Our work will enhance ongoing collaborations on ribosome production with internationally recognised experts such as David Tollervey (Edinburgh) and Markus Bohnsack (Göttingen, Germany). This project will also enable us to start new collaborations with researchers such as Alan Warren (Cambridge), looking at disease models, and Anne Willis (Leicester) to study changes in translation in cells in which ribosome production is disrupted.
5) TRAINING: We will provide excellent research training for the PDRA in a range of techniques used to analyse ribosome production, as well as specialised approaches, e.g. crosslinking studies and high-throughput sequencing. Training in a wide range of transferable skills, including presenting research at conferences and communicating science to the public, will also be provided to enhance the future career prospects of the PDRA.