Alternative splicing in the regulation of planarian stem cells in vivo: a conserved CELF/MBNL antagonism regulates stem cell self-renewal and differentiation.

• Applicant: Dr. Jordi Solana García
• Subject Area: Developmental Biology ; Evolutionary Cell and Developmental Biology (Zoology)
• Term: from 2014 to 2017
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 256258154

Alternative splicing (AS) is the process by which alternative exons are selectively included or excluded in the mature mRNA to produce multiple mRNA and protein isoforms, often with different functions. In recent years, deep sequencing techniques have enabled transcriptome-wide studies and substantially boosted research of alternative splicing. Several lines of evidence show AS being an essential process for the pluripotency network that regulates stem cells. However, the field lacks a transcriptome-wide perspective of AS in stem cells in vivo. Freshwater planarians offer an excellent platform for in vivo stem cell research. Their unique stem cells -the so-called neoblasts- underlie their amazing power of regeneration. Neoblasts are pluripotent, can be isolated in large numbers and easily manipulated by loss of function techniques. Recent transcriptomic studies from the Rajewsky laboratory uncovered that there is a deep conservation of the mechanisms governing pluripotency between planarian and mammalian stem cells. Thus, planarians are an informative in vivo model for human stem cell biology. We have identified and experimentally validated planarian stem cell-specific AS exons, and studied putative tissue specific AS factors in planarians (unpublished, preliminary data). These experiments have revealed that only two kinds of AS factors -CELF and MBNL factors- exert most of this regulation and antagonize to regulate AS in stem cells. CELF and MBNL factors act promoting and repressing the stem cell-specific AS respectively, by affecting alternative exon inclusion levels in opposing ways. Furthermore, the role of MBNL factors has been recently shown to be conserved in human stem cells, while the antagonism of human CELF factors in this process has only been sketched. These observations therefore point to a scenario in which AS is a key conserved process for regulating stem cells, in both mammalian and planarian stem cells. To expand our knowledge on how this antagonism regulates stem cells I want to characterize the functional consequences of CELF and MBNL loss-of-function in planarian stem cells and to elucidate their direct RNA binding targets. I will characterize the cellular effects of CELF and MBNL RNAi knock down on stem cell maintenance or differentiation and planarian regeneration and the molecular effects on alternative splicing. I also aim at developing in vivo PAR-CLIP in planarians, a method that allows the transcriptome-wide identification of RNA binding sites at nucleotide resolution. Integration of functional and RNA binding data will reveal the functional consequences of the conserved CELF/MBNL antagonism for stemness or pluripotency in vivo and characterize its mechanism of action in mRNA regulation. This research project will generate insights into how post-transcriptional mechanisms regulate stem cells in vivo and provide mechanistic information on the process, as well as on its evolutionary conservation.

• DFG Programme: Research Grants