Photocatalytic Chemical Crosslinking for Profiling RNA-Protein Interactions in Living Cells.
Huixin LuoWei TangHongyu LiuXiangmei ZengWilliam Shu Ching NgaiRui GaoHeyun LiRan LiHuangtao ZhengJianting GuoFangfei QinGang WangKexin LiXinyuan FanPeng ZouPeng R ChenPublished in: Angewandte Chemie (International ed. in English) (2022)
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.
Keyphrases
- mass spectrometry
- single cell
- induced apoptosis
- living cells
- sars cov
- cell cycle arrest
- multiple sclerosis
- visible light
- endothelial cells
- fluorescent probe
- liquid chromatography
- nucleic acid
- ms ms
- protein protein
- highly efficient
- rna seq
- binding protein
- endoplasmic reticulum stress
- adipose tissue
- signaling pathway
- high performance liquid chromatography
- high resolution
- inflammatory response
- gene expression
- gas chromatography
- cell death
- respiratory syndrome coronavirus
- protein kinase
- tandem mass spectrometry