Genome-wide translational responses to stress: a focus on initiation

Our bodies are made of very different types of cells: Skin cells are flat and protect our body, while brain cells have cables that pass messages around. Despite being so different, all our cells carry exactly the same information in their genes. What makes them special is what information they use, that is, which genes they switch on and off.

Cells need to respond to changes in their environment (stress) to avoid damage or even death. Stress conditions include high or low temperatures, lack of nutrients or a poor supply of oxygen. Cells react to stress by varying the way in which they use the information from their genes.

The information on how to make a cell is stored in the form of a DNA molecule. However, this information cannot be read directly: it first needs to be copied into another molecule called messenger RNA (mRNA), from which it can be 'translated' into a protein. Proteins are the components that directly build the cell and make it function, and it is also proteins that are responsible for protecting the cell from the damage caused by stress.

Cells react to stress by switching on 'defence' genes and by switching off the genes that are not needed during the response to stress. The process of turning on and off genes often takes place at the level of the translation of messenger RNAs (that is, by selecting which messenger RNAs will be translated into proteins). Translation is performed by tiny machines within the cells called ribosomes. Studying translation is relevant for human cells, because the mechanisms that regulate translation often go awry during cancer and several inherited conditions.

Our aim is to understand how cells change the information they use - especially through translation - to cope with situations of stress. Two questions are particularly important: where do ribosomes start reading the messenger RNA? How frequently do they start reading it? This information is crucial, because it determines how much of the protein is produced through translation of the messenger RNA - and therefore whether the gene will be switched on or not.

A recently-developed experimental technique allows us to detect all ribosomes on messenger RNAs as they prepare for the process of translation, giving us information about how translation changes in different situations. We will apply this method to study how cells modify translation of messenger RNAs in response to several stress conditions, and to understand how these changes help cells survive.

One way to study a complicated process of the human body is to use a model organism: this is a simpler creature, but similar enough to allow us to learn about ourselves. To study these questions we will employ a simple yeast -made of a single cell- that can react to many different types of stress. We will investigate how the yeast cells regulate translation in response to stress: which mechanisms they use, which genes are turned on an off, and what is the importance of these genes.

We expect this information will be useful to understand how human cells behave and, eventually, help us devise cures for disease.

Translation has been extensively studied at the genome-wide level using ribosome profiling, which allows the quantitative mapping of ribosomes on mRNAs. However, this approach only detects complete ribosomes, and thus cannot be used to examine the behaviour of small ribosomal subunits (SSUs), which are crucial for translation initiation. A novel approach, called 'Translation Complex Profiling' (TCP-seq), allows the systematic detection of both SSUs and full ribosomes on mRNAs, providing a complete and systematic picture of translation initiation. Despite its potential, TCP-seq has only been applied to one organism in a single condition.

We will use the fission yeast Schizosaccharomyces pombe to study how cells remodel translation initiation upon stress. We will apply TCP-seq (to map complete ribosomes and SSUs) to S. pombe control and stressed cells. This will be done in wild type cells and in mutants with an impaired stress response. We will complement these genome-wide studies with a detailed molecular investigation of the control of translation initiation of a key regulator of stress responses (fil1).

The TCP-seq experiments will shed light on the behaviour of SSUs during translation initiation, revealing global properties and transcript-specific features. The results will be compared with published datasets (currently only Saccharomyces cerevisiae) to identify general and species-specific regulatory principles. This work will also illuminate the mechanisms that target initiation upon stress, both to regulate global translation and specific transcripts. Finally, we will gain insight into the translational control of fil1, a new model system to study stress-regulated control of initiation.

This project will provide a comprehensive view of the molecular mechanisms and regulation of translation initiation in unstressed and stressed cells. We expect that this work will allow the discovery of general principles that may be applicable to human cells.

This project will contribute to the training of researchers in key areas of research, and will result in knowledge that may have long-term implications in various areas of medical research as described below.

The biotechnology and pharmaceutical industries are potential beneficiaries of this project, through the training of highly qualified researchers (point 1) and the knowledge and expertise it will generate (points 2 and 3). In addition, the project may contribute to fighting human disease, which would benefit the general public (points 2 and 3). The 'Pathways to Impact' document discusses in detail how we will ensure that all potential beneficiaries will be reached.

1] Training and capacity building in functional genomics / systems biology. Although the researcher co-investigator is already experienced in experimental methods, this project will provide an excellent opportunity for training in state-of-the-art analysis of large scale datasets (including the acquisition of programming skills). This will be done through work in our laboratory, as well as courses and interactions with members of the Systems Biology Centre. The provision of scientists trained in these multidisciplinary approaches will be beneficial for the UK industry, especially the biotechnology and pharmaceutical sectors. Indeed, two former postdocs of my laboratory (including one who worked on a BBSRC-funded project) moved to UK Biotechnology companies after leaving my lab. This is a key objective of the BBSRC Strategic Plan Enabling Theme 1 'Enabling innovation', which states that 'as bioscience becomes increasingly quantitative, there is also an urgent need to raise the mathematical and computational skills of biologists at all levels', and defines as a key priority 'training of bioscience researchers, particularly around our three strategic priorities and in the development of mathematical and computational skills'. Training in new technologies is also a key priority of Enabling Theme 2 'Exploiting new ways of working', which aims to 'enhance skills and capacity to exploit new tools and approaches e.g. through training for researchers'.

2] General understanding of human disease: Defects in the regulation of translation have been implicated in human diseases including cancer (1). For example, many initiation factors are overexpressed in cancer tissues, and their increased expression correlates with malignancy and poor prognosis (2). Moreover, numerous inherited syndromes are due to mutations in genes encoding translation factors or regulators of translation, as well as to mutations that alter RNA regulatory sequences (such as uORFs or binding sites of RNA-binding proteins) (3). This proposal aims at identifying general principles of how translation is regulated, which may be applicable to human cells.

3] As described in the 'Academic Beneficiaries' section, recent work has shown similarities between pathogens of the Pneumocystis genus and fission yeast (in particular, in their meiotic pathways). These organisms cause pneumonia in patients with weakened immune systems (premature babies, AIDS and cancer patients). As Pneumocystis cannot be cultured in vitro, there is a need for model systems that allow the study of their basic biology. As stress responses are essential for the survival of microorganisms, our results might be useful to understand the biology of these pathogens and develop treatments against their infection. To ensure this information reaches the Pneumocystis research community, we will highlight these similarities in peer-reviewed publications and relevant scientific conferences.

(1) Tahmasebi et al. 2018 'Translation deregulation in human disease', Nat Rev Mol Cell Biol doi: 10.1038/s41580-018-0034-x
(2) Ruggiero 2013 'Translational control in cancer etiology', Cold Spring Harb Perspect Biol 1;5(2)
(3) Scheper et al. 2007 'Translation matters: protein synthesis defects in inherited disease', Nat Rev Genet 8 711