RT Journal Article SR Electronic T1 The kinetic landscape of an RNA binding protein in cells JF bioRxiv FD Cold Spring Harbor Laboratory SP 2020.05.11.089102 DO 10.1101/2020.05.11.089102 A1 Sharma, Deepak A1 Zagore, Leah L. A1 Brister, Matthew M. A1 Ye, Xuan A1 Crespo-Hernández, Carlos E. A1 Licatalosi, Donny D. A1 Jankowsky, Eckhard YR 2020 UL http://biorxiv.org/content/early/2020/05/13/2020.05.11.089102.abstract 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.