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Profiling RNA subcellular localization in situ by TATA-seq

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE287794 | GEO | 2025/09/24

REPOSITORIES: GEO

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Project description:Introduction:Repetitive RNA sequences like CAG repeats that trigger aberrant RNA-protein interactions cause several neurodegenerative diseases including Huntington's disease (HD). Importantly, these aberrant RNA-protein complexes also seed the formation of cytoplasmic liquid-like granules, such as the stress granules. Emerging evidence demonstrates that granules formed via liquid-liquid phase separation can mature into gel-like inclusions that persist within the cell and may act as precursor to aggregates that occur in patients’ tissue. Thus, deregulation of RNA granule biology is an important component of neurodegeneration. Interestingly, the formation of intracellular membrane-less organelles like stress granules increases along with the secretion of exosomes. Exosomes are small membrane-bound vesicles that are secreted generally by all cell types. They may participate in the spreading of misfolded proteins and aberrant RNA-protein complexes across the central nervous system in neurodegenerative diseases like HD. Moreover, due to their capability to cross the blood-brain barrier, exosomes hold great potential as sources of biomarkers available from the periphery, e.g., from blood. In this study, we performed a comparative transcriptomic analysis of exosomes and RNA granules in an HD model.Methods: Exosomes and RNA granules were isolated from an HD cell model, stably expressing HTT exon 1 with 83 CAG repeats under an inducible promoter. Both exosomes and RNA granules were isolated from induced (HD) and non-induced (control) cells and analysed by RNA sequencing.Results: Comparative analysis between the transcriptomics data of HD RNA granules and exosomes showed that: (I) intracellular RNA granules and extracellular RNA vesicles share content, (II) several non-coding RNAs translocate to RNA granules, (III) the composition of RNA granules and exosomes is affected by expression of mutant HTT.Discussion: Our data show that intracellular RNA granules and exosomes share content, suggesting that formation of RNA granules and exosome loading may be related. Moreover, our data suggest that both intracellular RNA granules and extracellular RNA vesicles may serve as a source for diagnostic strategies. For example, disease-specific RNA-signatures of exosomes can serve as biomarker of central nervous system diseases. However, the cell-type specific signature of EV-secreted RNAs may make it difficult to detect these biomarkers in blood.

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Profiling RNA subcellular localization in situ by TATA-seq

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