How does nonsense-mediated decay contribute to gene regulation in developing neurons? (360G-Wellcome-211848_Z_18_Z)

Nonsense mediated decay (NMD) is a quality control mechanism used by eukaryotic cells to destroy messenger RNAs containing incorrectly positioned translation termination codons [1]. NMD, in combination with alternative RNA splicing (AS), also provides a potent mechanism for natural changes in gene expression in developing brain [2-5]. The main goal of my summer project will be to test the hypothesis that progressive down-regulation of an RNA-binding protein called PTBP1 during mammalian neurogenesis promotes neuronal identity by changing AS patterns and triggering NMD of multiple neural precursor-specific transcripts. I will first follow up on the RNA sequencing screen carried out in the Makeyev lab and validate bioinformatically predicted AS-NMD targets using Reverse Transcription PCR and quantitative PCR analyses of developing nervous system samples and embryonic stem cells undergoing neuronal differentiation in vitro. I will then analyse AS-MND regulation for one example showing the most robust regulation. This will be achieved by designing minigene and CRISPR-Cas constructs specific for AS-NMD promoting exons and testing these reagents in mouse ES cells undergoing neuronal differentiation or treated with siRNA against PTBP1. The results of this work should improve our understanding of the AS-NMD pathway and evaluate its contribution to neuronal differentiation.