Chen, Y.; Liu, Z.; Wang, Y.; Lu, W.; Li, H.; Wang, W.; Qiu, Z.; Qiu, Y.; Qing, H.; Xie, Y.; Liu, N.; Zhang, C.; Chen, Y.; Qin, W.

RNA and proteins interact pervasively in cellular processes, yet the functional relevance of most RNA-binding proteins remains unknown. To address this, we developed RNA-dependent thermal proteome profiling (RTPP), a method that identifies RNA-regulated proteins (RRPs) by detecting changes in structural stability of a protein upon RNA binding, without requiring crosslinking or enrichment. Applying RTPP in HEK293T cells revealed 1664 RRPs, including 257 previously undetected RNA-binding orphans. One such orphan, SGK3, binds the lncRNA CASC15, facilitating its PtdIns(3)P binding, endosomal recruitment and kinase activity. We further generated a tissue-specific RRP atlas, identifying interactions unique to particular organs, including hippocampus-specific RRPs dysregulated in Alzheimer's disease. Integrating RTPP with proximity labeling resolved RNA-binding heterogeneities across subcellular compartments. Live-cell RNase treatment also uncovered proteins associated with cell surface RNA, revealing that RO60 form nanoscale clusters with glycoRNA. RTPP provides a powerful approach to decipher functional RNA-protein interactions across multiple biological dimensions.