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Identification of alternative splicing networks defining embryonic stem cell identity

Final Report Summary - SPLICE-STEM (Identification of alternative splicing networks defining embryonic stem cell identity)

Overview of the results
The SPLICE-STEM project aims at investigating the role of proteins controlling gene expression, more precisely the expression of RNA molecules which are produced from the genomic DNA, and their impact on the maintenance of stem cell properties and identity. During their maturation processes, RNAs are heavily modified by proteins that bind to them: the RNA binding proteins. In order to study the role of RNA binding protein (RBP) in stem cell fate, we studied mouse embryonic stem cells (ESC) cultivated in vitro.
Although we initially wanted to focus on protein involved in a mechanism called alternative splicing, our data pointed out that several RNA binding proteins encoding ribosomal proteins (RP) and ribosome associated proteins (RAP) are highly expressed in ESC but not in their daughter cells called differentiated cells. Ribosomes are the molecular machinery producing proteins from RNAs. Based on the available scientific knowledge 4 years ago, this observation felt very important and I decided to focus our project on these RNA binding proteins specific to ribosomes and to further decipher the role of these proteins in controlling ESC physiology. 3 on-going research programs emerged from the Marie Curie CIG grant:
1- Identification of a new ribosome biogenesis protein in ESC (project of a PhD student)
Marion Bruelle identified a novel protein involved in the assembly of ribosomes. She demonstrated that this protein is highly expressed in ESC and rapidly lost has they change identity to differentiate, for example into muscle type cells in vitro. Marion also established that this new factor is required for stem cell to keep their naïve identity but is dispensable for their differentiation. Finally, combining loos of expression assays and biochemistry, she demonstrated that this factor is required for the maturation for the large ribosomal subunit and efficient translation in ESCs. Therefor this project demonstrate that ribosomes are synthetized using different factors in stem cells (Article in preparation) and now raise the question of whether this has an impact on the molecular composition of ribosomes, a highly relevant question in the ribosome field.
2- Caracterization of ribosome composition plasticity in ESC (project of a permanent researcher). Following up on Marion Bruelle PhD results and their potential consequences on ribosome composition and function, Sebastien Durand initiated a research project combining transcriptomics and proteomics approaches to compare the protein composition of ribosomes between parental ESC and the differentiated progenies. He identified several ribosomal proteins that specifically present in ESC ribosomes and potentially impact their structure and translation capacities. He has developed specific antibodies against these factors and is currently deciphering their precise regulation and function in ribosomes and stem cell physiology.
3- Identification of RNA binding proteins enriched in stem cells responsible for aggressive brain cancers.
Nabila Berabez, a PhD student, has conducted a transcriptomic analysis to identify RNA binding proteins preferentially expressed in cancer stem cells responsible for aggressive brain tumors: glioblastomas. She identified 2 candidate proteins and she is currently describing their expression profiles in cancer stem cells as well as in human glioblastoma tumor samples. Moreover she developed a strategy to manipulate their expression in order to decipher their molecular functions in controlling gene expression in cancer stem cells, as well as to establish their importance for cancer stem cell survival. This is a key issue in the field of cancer treatment as many cancer types relapse after treatment, and it has been shown that cancer stem cells play an active role in this relapse (Article and thesis defense in preparation).
In conclusion, this CIG grant has enabled the identification of novel RNA binding proteins affecting alternative splicing decisions and impacting stem cell behaviour in vitro. It also unravelled a novel level of regulation affecting directly the translation machinery (ribosomes) in ESC, two projects currently being developed in my laboratory.

The perspectives of team are to concomitantly further develop our fundamental research programs focused on translation control of ESC fate and our applied research programs on cancer stem cells. We have identified several genes of interest in each models and we will continue to describe their molecular function as a first priority. In addition, we will develop novel strategies to control, alter or destroy their expression or functions, in particular in cancer stem cells, as a mean to propose novel innovative treatments to treat aggressive adult and pediatric brain tumors, including glioblastoma. We will benefit from the C3D drug discovery platform of the CRCL to first select chemical compounds interfering with our candidate proteins, and then to improve this targeting by modifying the candidate molecules.
To reach these objectives we are currently developing multidisciplinary approaches including biophysics, bioinformatics and imaging to better understand the function of our candidate factors. These technologies will further be developed in order to adapt the scale or the precisions of the questions we will be asking in the coming years. This will be achieved with our collaborators in biophysics from the University of Lyon (France), and with collaborators in computer science from the French National Institute for computer science and applied mathematics (INRIA) in Nice (France).

Socio-economic impact of the project.
The Marie-Curie CIG grant has allowed me to establish as a junior principal investigator as I returned from a postdoctoral training in Canada and obtained a permanent researcher position in the Cancer Research Centre of Lyon in France. In addition to the support it provided to develop our research programs, the CIG grant also provided me with a unique opportunity to hire a motivated PhD student and to attract two talented postdoctoral fellows who respectively obtained a assistant professor position in the University of Nimes (France) and a permanent researcher position from the french National Institute for Health and Medical Research (INSERM) in my laboratory. Importantly, we also established privileged discussions with 3 clinicians (a Neuro-oncologist and two neuro-pathologists) from the general hospital of Lyon who are directly implicated in developing the cancer related projects of the laboratory.
In addition, the Marie Curie CIG grant help me to secure several additional funding from local associations and entities, and also 3 competitive grants from National funding agencies, including one from the INSERM and 2 multi-team project I coordinate from the National Institute of Cancer (INCa). As part of establishing the laboratory, we also setup a collaborative network with several laboratories within the CRCL, in local research centres and national research Centres in the cities of Paris, Nice, Grenoble, Tours and Nancy. In the future, our objective is to extend this collaborative network to international collaborations, with a particular focus in laboratories within the EU.
In the past 4 years we have trained over 10 students to research, including 5 Master students and 3 M.D. students and 2 international students from the University of Leicester (UK) and the University of Beijing (China).
We currently have 2 papers in preparation, one for each main axis of the laboratory, which will highlight the work of two PhD students. A third publication in collaboration with a clinician from the lab is also in preparation.