The kinetic landscape of an RNA-binding protein in cells

Gene expression in higher eukaryotic cells orchestrates interactions between thousands of RNA-binding proteins (RBPs) and tens of thousands of RNAs1. The kinetics by which RBPs bind to and dissociate from their RNA sites are critical for the coordination of cellular RNA–protein interactions2. However, these kinetic parameters have not been experimentally measured in cells. Here we show that time-resolved RNA–protein cross-linking with a pulsed femtosecond ultraviolet 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 cross-linking and immunoprecipitation (KIN-CLIP) approach reveals that DAZL resides at individual binding sites for time periods of only seconds or shorter, whereas the binding sites remain DAZL-free for markedly longer. The data also indicate that DAZL bin

ds to many RNAs in clusters of multiple proximal sites. The effect 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 kinetic parameters for RNA–protein interactions can be measured in cells, and how these data link RBP–RNA binding to the cellular function of RBPs.

Time-resolved RNA-protein cross-linking with a fs laser

Sharma, D., Zagore, L.L., Brister, M.M. et al. The kinetic landscape of an RNA-binding protein in cells. Nature (2021).

Leave a Reply

Your email address will not be published. Required fields are marked *