Interconnection between R-loops and co-transcriptional alternative splicing

ended

R-loops are long RNA/DNA hybrids naturally formed behind RNA polymerase II (Pol II) during transcription, but their persistence is a threat for genome integrity through the creation of DNA damage, leading to cancer development. However, several recent studies reported a new and exciting role for R-loops in gene expression regulation by influencing transcription termination and chromatin modification. As mutations in several RNA processing factors have been shown to stabilize R-loop formation, we propose here to investigate the link between R-loops and co-transcriptional alternative splicing (AS) and to decipher the underlying mechanisms, a hypothesis supported by our preliminary results identifying more than 3000 splice junctions affected by decreased R-loops upon overexpression of RNase H1. The use of a specific antibody to RNA/DNA hybrids and stabilization/removal of R-loops coupled to high-throughput analysis of splicing (RNA-seq), R-loop profiling (DRIP-seq) and Pol II positioning and measurements (PRO-seq) will allow us to investigate genome-wide regulation of AS by R-loop formation. We will use particular AS events as models to elucidate the molecular mechanisms interconnecting R-loops and AS. Moreover, based on our previous expertise, we plan to study the impact of R-loops on DNA damage-induced AS alteration as a model of physiological regulation of AS by R-loops. This project will provide new insights on AS regulation through the formation of R-loops and also on cancer progression through R-loop stabilization. Finally, this project will allow me to acquire independent thinking and scientific leadership to reach an independent academic position in France.