FISH+ is a ready-to-use proximity labeling method for simultaneous RNA visualization and RNA-interacting protein identification.

Traditional proximity biotinylation approaches require extensive genetic engineering or intricate purification steps. Here, we introduce FISH+, a ready-to-use RNA proximity labeling method that relies on the recruitment of peroxidase to RNA targets, and facilitates in situ proximity biotinylation in fixed cells. This method permits concurrent visualization of RNA molecules and identification of RNA-interacting proteins. Using this method, we visualized 45S and NEAT1 RNA, and captured their proximal proteins, demonstrating the capability to concurrently visualize RNA and identify proximal proteins. By targeting PNCTR (~36 copies per cell), we observed distinctly bright RNA dots, representing the combined biotinylation signals from both RNA and proximal proteins in situ. This indicates the potential of the FISH+ method for enhanced RNA visualization. We further generalized the FISH+ method to explore XIST-interacting proteins, a number of reported interactors were significantly enriched, such as SPEN, CIZ1 and RBM15. Using quantitative mass spectrometry, we show that FISH+ correctly identifies known RNA-protein interactions in the nucleus of human cells. Overall, we established a watch-and-catch punctate RNA method through the integration of RNA fluorescence in situ hybridization (FISH) with proximity biotinylation. This method provides additional spatial information for the characterization of RNA-centric interactions in fixed, genetically unperturbed samples.