The multifunctional FUS protein: Characterization of the biological effects of its depletion

Ruepp, Marc-David (25 January 2013). The multifunctional FUS protein: Characterization of the biological effects of its depletion (Unpublished). In: Swiss RNA Workshop 2013. Bern, CH. 25.01.2013.

FUS/TLS (fused in sarcoma/translocated in liposarcoma) protein, a ubiquitously expressed RNA-binding protein, has been linked to a variety of cellular processes, such as RNA metabolism, microRNA biogenesis and DNA repair. However, the precise role of FUS protein remains unclear. Recently, FUS has been linked to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Based on the observation that some mutations in the FUS gene induce cytoplasmic accumulation of FUS aggregates, we decided to explore a loss-of-function situation (i.e. inhibition of FUS’ nuclear function) to unravel the role of this protein. To this purpose, we have generated a SH-SY5Y human neuroblastoma cell line which expresses a doxycycline induced shRNA targeting FUS and that specifically depletes the protein. In order to characterize this cell line, we have performed a whole transcriptome analysis by RNA deep sequencing. Preliminary results show that FUS depletion affects both expression and alternative splicing levels of several RNAs. When FUS is depleted we observed 330 downregulated and 81 upregulated genes. We also found that 395 splicing isoforms were downregulated, while 426 were upregulated. Currently, we are focusing our attention on the pathways which are mostly affected by FUS depletion. In addition, to further characterize the FUS-depleted cell line we have performed growth proliferation and survival assays. From these experiments emerge that FUS-depleted cells display growth proliferation alteration. In order to explain this observation, we have tested different hypothesis (e.g. apoptosis, senescence or slow-down growth). We observed that FUS-depleted cells growth slower than controls. Currently, we are looking for putative candidate targets causing this phenotype. Finally, since MEFs and B-lymphocytes derived from FUS knockdown mice display major sensitivity to ionizing radiation and chromosomal aberrations [1,2], we are exploring the effects of DNA damage in FUS-depleted cells by monitoring important components of DNA Damage Response (DDR). Taken together, these studies may contribute to our knowledge of the role of FUS in these cellular processes and will allow us to draw a clearer picture of mechanisms of neurodegenerative diseases.

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