Profiling RNA subcellular localization in situ by TATA-seq

Abstract

Membrane-less organelles, dynamic subcellular structures formed by RNA and RNA-binding proteins (RBPs) undergoing liquid–liquid phase separation (LLPS), play key roles in biological processes such as RNA degradation in processing bodies (P-bodies), translation inhibition in stress granules, and RNA splicing in nuclear speckles. However, the study of RNA species within these organelles has been hindered by the absence of simple, sensitive, and specific methodologies. Here, we introduce target transcript amplification and sequencing (TATA-seq), a novel strategy for precisely profiling RNA in membrane-less organelles via in situ targeted transcription and linear amplification. TATA-seq uses a primary antibody against a marker protein of the target organelle to recruit a secondary antibody conjugated with streptavidin, which binds an oligonucleotide containing a T7 promoter. This initiates in situ RNA reverse transcription, followed by amplification with T7 RNA polymerase to generate sufficient material for sequencing, ensuring a duplication rate of no more than 25% and a mapping ratio of ∼90%. An IgG control is used to subtract background noise during data analysis. We demonstrate the method's utility by profiling RNA in stress granules induced by sodium arsenite in HeLa cells, with validation through FISH and immunofluorescence colocalization. TATA-seq offers a simple, highly sensitive, and accurate tool for studying RNA dynamics in membrane-less organelles, advancing the capabilities of RNA research.