The kinetic landscape of an RNA binding protein in cells

The binding and dissociation of RNA Binding Proteins (RBPs) to their cognate sites on cellular RNAs are critical for the regulation of gene expression. Yet, association and dissociation kinetics of RBPs at individual cellular binding sites have not been experimentally accessible. Inaccessibility of binding and dissociation kinetics of RBPs in cells severely limits or even precludes the establishment of quantitative connections between RBP-RNA interactions and cellular RBP function. Here, we describe an approach to measure binding and dissociation kinetics of the RBP Dazl at thousands of individual binding sites in cells. We then show how these kinetic parameters inform a quantitative understanding of the cellular function of Dazl. Overall design: 16 iCLIP libraries corresponding to 4 time points, 2 in vivo Dazl concentrations and 2 femtosecond, pulsed UV laser powers. Monoclonal cell line with doxycyline inducible Dazl expression (GC-1-spg parental line) was utilized.