The work on this project started with the analysis of previously published data. I then developed and performed experiments to test our initial hypothesis and used the results obtained to extend and refine the scope of our study. Briefly, I analysed transcriptome-wide changes in mouse neuroblastoma cells treated with siRNAs against Ptbp1, and/or the translational inhibitor cycloheximide (CHX; also known to repress NMD) and identified a subset of novel Ptbp1-regulated AS-NMD targets encoding known actin cytoskeleton assembly factors. Analysis of longitudinal RNA-Seq data of in vitro differentiated mouse ESCs into glutamatergic neurons (Hubbard K.S. et al., 2013) confirmed that these targets are naturally downregulated during neuronal differentiation with kinetics similar to that of Ptbp1. I then performed in vitro differentiation of ESCs into neurons and validated the gene downregulation effect and increased inclusion of NMD-inducing (“poison”) exons for several targets. I also confirmed that the inclusion of the poison exon was indeed Ptbp1-dependent, and that it had a direct impact on downregulation of these targets at both mRNA and protein levels.
To gain further insights into the extent of Ptbp1 target repertoire, I established a mouse ESC line (mESC-Ptbp1-AID) that allows for rapid degradation (≃4h) of Ptbp1 using an auxin-inducible depletion system (Natsume T. et al, 2016). I performed several high-throughput experiments with this innovative Ptbp1 downregulation system and identified a previously unknown wider role for Ptbp1 in co-transcriptional intron removal. These experiments also indicated a novel mechanism of how this activity might be coupled with regulation of at least a subset of Ptbp1-controlled alternative exons. Interestingly, examples of the newly identified targets included genes required for the maintenance of the stem cell status and, again, normal assembly of the actin cytoskeleton.
All the genome-wide data generated with the mESC-Ptbp1-AID and the cell line itself provide a valuable community resource, since Ptbp1 is a pivotal regulator of alternative splicing and gene expression in a variety of normal and disease-associated biological contexts. Moreover, we have one manuscript ready for submission and another manuscript currently in preparation to be submitted to peer-reviewed journals.
As part of the dissemination of this project, I presented this research data in several international conferences such as the 2018 RNA society meeting in Berkeley, USA, the 2019 RNA society meeting in Krakow, Poland, and the EMBO workshop “RNP network dynamics in development and disease” in Ljubljana, Slovenia. I also joined outreach events such as the ‘British science week’ and the CDN summer school ‘DevNeuro academy’, which allowed me to discuss my research with students interested in undertaking scientific careers.
