TAS-seq enables subcellular single-stranded adenosine profiling by signal peptide-assisted adenosine deamination

RNA structure critically facilitates RNA function and undergoes alterations from transcription into its ultimate functional destination in different subcellular compartments. To enhance our understanding of RNA structural dynamics, we develop TAS-seq, which locates deaminase TadA-8e to specific subcellular compartments and modifies adenosine in single-strand structures, particularly within the hairpin loop, to profile the RNA secondary structure of subcellular RNAs. We identified variations across three subcellular components (nucleus, cytoplasm, and endoplasmic reticulum membrane) and two cell lines (HEK293T and K562), and investigated the interplay between RNA structural changes and RBP binding. We also performed TAS-seq at the single-cell level, achieving the first full-length RNA structure heterogeneity profiling of individual cells. Overall design: To profile the structure of subcellular RNAs, we expressed adenosine deaminase TadA-8e in human cells. TadA-8e is anchored into subcellular compartments (nucleus, cytoplasm, and endoplasmic reticulum membrane) by signal peptides (NLS, NES, and ERM). Then the total RNAs, poly(A) RNAs, and Ribo-RNAs were extracted and sequenced, separately. Adenosines in specific RNA structures were modified by TadA-8e resulting in A-to-G substitution in high-throughput data.