Profiling RNA subcellular localization in situ by TATA-seq
Membranelles 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 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 membranelles 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 with a ds-T7 promoter. This initiates in situ transcription of RNA transcripts, followed by amplification with T7 RNA polymerase to generate sufficient material for sequencing. 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. TATA-seq offers a simple, highly sensitive, and precise tool for studying RNA dynamics within membranelles organelles, expanding the capabilities of RNA research. Overall design: We performed TATA-Seq on sodium arsenite-treated HeLa cells to detect RNAs within stress granules. Both the AS-IgG and Ctrl-IgG groups employed the IgG antibody homologous to G3BP1 (the marker protein of stress granules) to help filter out background signals from non-specific binding. Both the KO-IgG and WT-IgG groups employed the IgG antibody homologous to TNRC6A to help filter out background signals from non-specific binding.