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Molecular mechanisms of RBM5-mediated regulation of alternative splicing: impact on cell proliferation and apoptosis

Final Report Summary - RBM5 (Molecular mechanisms of RBM5-mediated regulation of alternative splicing: impact on cell proliferation and apoptosis)

1.a) Scientific project

1.a.1) Description and objectives

According to the world health organization (WHO), more than 11 million people are diagnosed with cancer every year. This number is rising and will amount to 16 million new cases by 2020. Cancer is one of the leading causes of death worldwide (12.5%) and in the EU. The European Cancer Leagues states that two million Europeans are diagnosed with cancer every year.

In addition, the European Cancer Patient Coalition states that In Europe, every day 5.214 Europeans are diagnosed with cancer and 3.185 die from their disease (20% of total cases are related to lung cancer). Understanding of how genetic factors contribute to these diseases is gathering speed. Genome-wide association analyses on large patient cohorts are generating large sets of candidate genes. This is coupled with the availability of ever-increasing genomic databases and a rapidly expanding repository of biomedical literature.

To investigate how defects in pre-mRNA processing could be a major cause in tumor formation and progression I focused on three related RNA Binding Proteins, RBM5, RBM6 and RBM10.

RBM5 and RBM6 genes map to chromosomal region 3p21.3 that is frequently deleted in heavy smokers, lung cancer and other carcinomas. RBM5 was shown to regulate alternative splicing of apoptosis related genes (Bonnal et al., 2008, Fushimi et al., 2008) and the three proteins are suggested to play important roles in tumor development and progression (Rintala- Maki et al., 2007). However, very little is known about the molecular functions of these proteins and their involvement in tumor formation.

1.a.2) Results

During my training I was able to combine technically challenging state of art genomewide technologies with detailed mechanistic studies to understand the role of these proteins in the control of cell proliferation. I successfully identified RNA targets of these factors in Hela cells using Cross Linking Immunoprecipitation coupled to deep sequencing (CLIP-Seq), which allows genome-wide identification of specific RNA/protein interactions. We showed that RBM5, six and ten targets are involved in signal transduction, cell division, proliferation and apoptosis pathways.

We also used human splicing sensitive microarrays after depletion of each protein and observed that expression and/or splicing of more than 1000 genes are affected, with both common and distinct targets for each protein. Combining both CLIP-seq and microarrays data we were able to reveal distinct functional RNA maps which allows to predict the splicing regulation outcome based upon the location of the factor's binding sites in the pre-mRNA. The genome wide analysis offered to us a vast repertoire of potential targets important for cell proliferation. Significantly, we could identify targets in which differential effects of these proteins can explain their distinct activities in the control of cell proliferation. Thus, we could show that NUMB exon 9 alternative splicing, a highly regulated splicing event in breast and lung cancer cell lines, is a key mediator of the effects of RBM proteins in cell growth, as manipulation of this event by silencing of endogenous exon 9 inclusion or ectopic expression of different NUMB isoforms recapitulates the different effects on proliferation effects caused by depletion of different RBM proteins. Our results therefore uncover a post-transcriptional regulatory circuit important for the control of cancer cells.

1.a.3) conclusions

We were able to demonstrate that, although they are highly identical, RBM5 RBM6 and RBM10 play different roles in regulating alternative splicing of cell proliferation and apoptosis related genes.

1.a.4) Socio-economical impact

As cancer diseases are one of the major causes of mortality in the population of the European countries, an important effort is required in order to improve their life quality. These goals should be achieved by ameliorating diagnosis, prevention as well as treatment. Therefore, our detailed study of the mechanisms of action of RBM5, RBM6 and RBM10 and their impact on cell proliferation and apoptosis, should allow us to conceive new tools to improve life quality.

We believe that the precise knowledge of these proteins could allow us the design of new potential therapeutic strategies aimed to inhibiting the massive cell transformation occurring during the tumor progression processes.