Directed evolution of Lantern enables rapid RNA and protein proximity labeling

Abstract

In eukaryotic cells, the precise spatial localization of RNAs and proteins is essential for proper cellular function. Genetically encoded photocatalytic proximity labeling techniques have expanded our ability to map subcellular proteomes and transcriptomes, but their temporal resolution remains limited. Here we introduce Lantern, an engineered flavoprotein optimized via directed evolution, which enables sub‑minute, spatially resolved labeling of cellular biomolecules. Lantern is targetable to diverse subcellular compartments, including the endoplasmic reticulum (ER), mitochondria and stress granules (SGs), to map local transcriptomes (CAP-seq) and proteomes (CAP-MS). Using Lantern, we observed that N6-methyladenosine-rich RNAs are recruited to SGs within 10 minutes of stress induction, and ER‑proximal RNAs associate with G3BP1 during early SG assembly. Additionally, Lantern was adapted for cell surface tagging (CAP-CELL), enabling spatially resolved cell typing and identifying cell−cell interactions. Collectively, this study establishes Lantern as a powerful tool that offers unprecedented temporal resolution for investigating the dynamic organization of subcellular molecular networks. Fang, Ren, Zheng, Wang et al. developed Lantern, a variant of miniSOG, through directed evolution for subcellular transcriptomes and proteomes analyses inside living cells, enabling findings that RNA mobilization precedes protein recruitment in stress granule assembly.