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 binds 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.

高等真核细胞中的基因表达协调了数千个 RNA 结合蛋白 (RBP) 和数万个 RNA 之间的相互作用¹。RBP 与其 RNA 位点结合和解离的动力学对于细胞 RNA-蛋白质相互作用的协调至关重要². 然而，这些动力学参数尚未在细胞中进行实验测量。在这里，我们展示了用脉冲飞秒紫外激光进行时间分辨 RNA-蛋白质交联，然后进行免疫沉淀和高通量测序，可以确定 RBP DAZL 对数千个单独 RNA 结合位点的结合和解离动力学细胞。这种动力学交联和免疫沉淀 (KIN-CLIP) 方法揭示了 DAZL 在单个结合位点的停留时间仅为几秒或更短，而结合位点保持无 DAZL 的时间明显更长。数据还表明 DAZL 与多个近端位点簇中的许多 RNA 结合。DAZL 对 mRNA 水平和核糖体结合的影响与 DAZL 在这些簇中结合的累积概率相关。将动力学数据与 mRNA 特征相结合，定量地将 DAZL-RNA 结合与 DAZL 功能联系起来。我们的结果显示了如何在细胞中测量 RNA-蛋白质相互作用的动力学参数，以及这些数据如何将 RBP-RNA 结合与 RBP 的细胞功能联系起来。