Longitudinal monitoring of cytoplasmic RBP-RNA interactions and transcriptome in living cells by engineered protein nanocages

Here, we introduce POND-seq (Protein nanocage-empOwered Non-Destructive sequencing), a strategy that employs secretory protein nanocages fused to RNA-binding proteins (RBPs) to recover RBP-associated RNAs from living cells. POND-seq robustly identifies RNA targets of cytoplasmic RBPs across multiple cell types and enables longitudinal tracking of dynamic changes in RBP-associated RNA profiles under stress conditions.

Fusion to PABPC1 further allows monitoring of transcriptomic responses and selectively profile cell-type-specific transcriptomes from mixed populations without cell dissociation and sorting. Additionally, POND-seq supports functional interrogation of RBP domains and residues involved in RNA association and enables scalable analysis of RBP variants, as demonstrated by a systematic assessment of disease-associated FMR1 mutations.