Photocatalytic Chemical Crosslinking for Profiling RNA–Protein Interactions in Living Cells
Huixin Luo, Wei Tang, Hongyu Liu, Xiangmei Zeng, William Shu Ching Ngai, Rui Gao, Heyun Li, Ran Li, Huangtao Zheng, Jianting Guo, Fangfei Qin, Gang Wang, Kexin Li, Xinyuan Fan, Peng Zou, Peng R. Chen
Abstract
The dynamic interactions between RNAs and proteins play crucial roles in regulating diverse cellular processes. Proteome-wide characterization of these interactions in their native cellular context remains desirable but challenging. Herein, we developed a photocatalytic crosslinking (PhotoCAX) strategy coupled with mass spectrometry (PhotoCAX-MS) and RNA sequencing (PhotoCAX-seq) for the study of the composition and dynamics of protein-RNA interactions. By integrating the blue light-triggered photocatalyst with a dual-functional RNA-protein crosslinker (RP-linker) and the phase separation-based enrichment strategy, PhotoCAX-MS revealed a total of 2044 RBPs in human HEK293 cells. We further employed PhotoCAX to investigate the dynamic change of RBPome in macrophage cells upon LPS-stimulation, as well as the identification of RBPs interacting directly with the 5' untranslated regions of SARS-CoV-2 RNA.