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

ABSTRACT: Most existing RNA sequencing methods rely on cell lysis or fixation, limiting their use in longitudinal studies of the same cell population. 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. Together, POND-seq provides a versatile and scalable platform for non-destructive and longitudinal analysis of cytoplasmic transcriptome and RBP-associated RNAs.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE293919 | GEO | 2026/06/24

REPOSITORIES: GEO

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