Structural and functional investigations on the consequences of Sam68 post-translational modifications on alternative splicing regulation

The identification of 20-25,000 human genes by the human genome project came as a big surprise since the estimated number of human proteins is around 130,000 and most scientists assumed a similar number of human genes. This discrepancy can only be explained if one single gene can generate many proteins. It then became clear that alternative RNA splicing is a major regulatory event in cells. Alternative splicing allows for the production of many messenger RNAs and consequently proteins from a single gene by splicing the precursor messenger RNA in many different ways. This process greatly increases the diversity of proteins in humans from a limited number of genes. It is highly regulated by RNA binding proteins, called splicing factors, and defects in its regulation lead to a large number of diseases, including genetic disorders and cancer, often due to overexpression or mutations of splicing factors.

Alternative splicing regulation is modulated by extracellular signals through cell signalling pathways that typically modify specific amino acids on target proteins to modulate their functions. A typical example is the splicing factor Sam68, which is overexpressed in a large number of cancers and whose function in alternative splicing is strongly modulated by signalling pathways. Many modifications of Sam68 in response to signalling pathways have been identified, such as phosphorylation, methylation and acetylation. However, very little is known about the molecular mechanisms that govern these regulations and modulations.

We have recently revealed the structure of Sam68 in complex with its RNA target and proposed a model for Sam68's mechanism of action in alternative splicing regulation.

In this proposal, we will further investigate the regulatory network involving Sam68 by analyzing the effects of cell signalling pathways on Sam68 alternative splicing functions. To that aim, we will address three complementary questions:

1- Which amino acids of Sam68 are modified in cells?
2- Which modifications of Sam68 occur in response to EGFR pathway, a pathway that is often associated with cancer progression?
3- What are the consequences of these modifications on Sam68 structure, RNA binding, localization and alternative splicing function?

This will allow us to (i) investigate the mechanism of action of each amino acid modifications on Sam68 structure, dynamic, localization, RNA binding and function, and (ii) evaluate the contribution of Sam68 in the EGFR signalling pathways that is often deregulated in diseases such as cancer.

Cell signalling and alternative splicing are tightly interconnected but the molecular basis of this relationship remains largely unknown. The oncogenic splicing factor Sam68 is known to provide a direct link between these events: Sam68 regulates alternative splicing of many pre-mRNAs encoding for oncogenic isoforms but its action in splicing regulation is highly modulated by signalling pathways through post-translational modifications, such as phosphorylation, methylation and acetylation. However, the precise mechanism of action of these modifications remains largely unknown.

Our aim is to decipher the regulatory networks of Sam68. We have already unraveled the structural basis of Sam68 dimerization and RNA binding, which allowed us to propose a model whereby Sam68 regulates alternative splicing by looping out regions of the pre-mRNA (Feracci, et al, Nat. Commun., 2016). In this proposal we will investigate how post-translational modifications affect Sam68 structure, localization, RNA binding and functions in alternative splicing. We will focus on the poorly understood Serine/Threonine phosphorylation of Sam68 and the EGFR signalling pathway in prostate and colorectal cancer cells and organoid cultures because both Sam68 and component of the EGFR pathway are frequently mutated in these types of cancer.
In recent years, Nuclear Magnetic Resonance (NMR) and quantitative Mass Spectrometry (MS) techniques have allowed PTMs of proteins to be monitored in real-time and at atomic resolution. We will use these two techniques in combination with biochemical and cell biology assays to address the following questions:
1- Which amino acids of Sam68 are modified in cells?
2- Which modifications of Sam68 occur in response to EGFR pathway?
3- What are the consequences of these modifications on Sam68 structure, RNA binding, localization and alternative splicing function?

Although cancer survival rates have significantly increased over the years thanks to basic and applied research outcomes, the fight against cancer is not over since certain types of cancers still display a poor survival rate, and drug resistance mechanisms developed by cancer cells remain a major issue for cancer research.
Sam68 is an oncogenic splicing factor whose overexpression in many types of cancers correlates with poor prognosis and there are strong evidence that this is at least partly due to Sam68's functions in alternative splicing. This suggests that inhibiting Sam68's functions should have a beneficial effect for the treatment of many types of cancer. Our research proposal therefore offers a unique opportunity to decipher the mechanisms of action of this oncogenic protein and develop novel therapeutic molecules. Thus, the outcome of our research will certainly have an economic and societal impact in the long term.

Economic and societal impact
Over the past few years, technological advances and increased knowledge in basic sciences have changed the views of the drug discovery community and a large number of targets previously considered as "undruggable" are increasingly reassessed by the chemical biology/drug discovery community. A typical example is the search for drugs that affect alternative splicing. To that aim, pharmaceutical companies have initiated research programs aimed at targeting alternative splicing deregulations in genetic diseases and cancers and small molecules or antisense oligonucleotides have entered clinical trials. We are therefore confident that a better understanding of the regulation of Sam68 functions in alternative splicing will attract their interest. This is highlighted by our ongoing collaboration with the European Lead Factory, a collaborative public-private partnership, aiming at identifying inhibitors of Sam68 RNA binding. In this proposal, we will investigate the contribution of Sam68 function in alternative splicing following cell treatment by well-known enzyme inhibitors, some of which are already approved drugs. This will undoubtedly increase our knowledge on the downstream effects of these drugs, which could be beneficial to reduce undesirable side effects of these drugs or better understand the mechanisms of resistance by cancer cells and will be used to develop novel targeted therapies that will be beneficial for patients suffering from cancer.

Academic impact
In this proposal, we will acquire the technology of in-cell NMR and transfer it to the UK through collaboration with Prof. Ito, a world leader in this technology. NMR analysis of proteins in cell extracts and in living cells allow for the first time to perform structural biology studies of macromolecules in a cellular context. We will further improve this technology by applying it to patient-derived cancer cells and organoid cultures, providing for the first time structural details of a protein in a disease-relevant physiological system.
The project will involve a young post-doctoral researcher and a research technician. We will guide and train them in research and lab management. The PDRA will be the main contributor of published research articles, enabling him to apply for competitive fellowships. He/she will participate in national and international conferences presenting posters and giving oral presentations describing his/her results. To develop his/her supervisory skills, he/she will be involved in guidance of MSc and Ph.D. students. To assist with these matters we have an implemented departmental mentoring system and yearly appraisals of PDRAs.