Tools4Cells: TimeCLIP technology to enable time-resolved detection of protein-RNA interactions

RNA-binding proteins help control when, where, and how genetic information is used within cells. Existing methods can identify contacts between proteins and RNA, but they generally provide only a single snapshot and therefore cannot determine when an interaction occurs during the lifetime of an RNA molecule. This project will establish timeCLIP as a technology for resolving protein–RNA interactions according to the age of the RNA, with a particular focus on extending the approach to proteins and RNA structures that are difficult to capture using current methods. By enabling temporal analysis of these challenging interactions, the project will provide broadly useful biotechnology for studying gene regulation, cellular function, and processes relevant to human health. Broader-impact activities will include graduate and postdoctoral training in RNA biology, genomics, quantitative analysis, and responsible research, together with dissemination of protocols and benchmark datasets to the scientific community. The project will establish timeCLIP for time-resolved mapping of protein–RNA interactions and address two major barriers to its application across diverse RNA-binding protein classes. First, alternative photoactivatable ribonucleosides and ultraviolet irradiation wavelengths will be evaluated to enable temporal mapping of RNA-binding proteins that crosslink inefficiently under standard conditions. Second, timeCLIP will be adapted for double-stranded RNA-binding proteins and protein-associated RNA–RNA hybrids by combining time-resolved metabolic labeling with intermolecular RNA ligation and dedicated computational analysis. Method development will use representative RNA-binding protein classes, matched input libraries, established CLIP datasets, and validation in relevant cellular systems. Performance will be evaluated for specificity, reproducibility, temporal discrimination, and recovery of established and newly detectable interactions. These studies will generate validated experimental workflows, benchmark datasets, and analysis strategies for resolving challenging protein–RNA interactions and RNA architectures. Together, these advances will establish timeCLIP as a versatile platform for investigating temporal RNA regulation across interaction classes that remain difficult to study with current approaches. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.