PT - JOURNAL ARTICLE AU - Sharma, Deepak AU - Zagore, Leah L. AU - Brister, Matthew M. AU - Ye, Xuan AU - Crespo-Hernández, Carlos E. AU - Licatalosi, Donny D. AU - Jankowsky, Eckhard TI - The kinetic landscape of an RNA binding protein in cells AID - 10.1101/2020.05.11.089102 DP - 2020 Jan 01 TA - bioRxiv PG - 2020.05.11.089102 4099 - http://biorxiv.org/content/early/2020/05/13/2020.05.11.089102.short 4100 - http://biorxiv.org/content/early/2020/05/13/2020.05.11.089102.full AB - Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA binding proteins (RBPs) and tens of thousands of RNAs 1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA-protein interactions 2. However, these kinetics were experimentally inaccessible in cells. Here we show that time-resolved RNA-protein crosslinking with a pulsed femtosecond UV laser, followed by immunoprecipitation and high throughput sequencing allows the determination of binding and dissociation kinetics of the RBP Dazl for thousands of individual RNA binding sites in cells. This kinetic crosslinking and immunoprecipitation (KIN-CLIP) approach reveals that Dazl resides at individual binding sites only seconds or shorter, while the sites remain Dazl-free markedly longer. The data further indicate that Dazl binds to many RNAs in clusters of multiple proximal sites. The impact of Dazl on mRNA levels and ribosome association correlates with the cumulative probability of Dazl binding in these clusters. Integrating kinetic data with mRNA features quantitatively connects Dazl-RNA binding to Dazl function. Our results show how previously inaccessible, kinetic parameters for RNA-protein interactions in cells can be measured and how these data quantitatively link RBP-RNA binding to cellular RBP function.Competing Interest StatementDeepak Sharma and Eckhard Jankowsky are founders of Bainom Inc.