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Updated: Sep 13, 2025

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Profiling RNA subcellular localization in situ by TATA-seq.

Junjie Li1, Chu Xu1, Xiao Jiang1

  • 1Cancer Institute, Fudan University Shanghai Cancer Center, Institutes of Biomedical Sciences, Shanghai Key Laboratory of Medical Epigenetics, International Co-laboratory of Medical Epigenetics and Metabolism (Ministry of Science and Technology), Shanghai Medical College of Fudan University, Shanghai 200032, China.

RNA (New York, N.Y.)
|August 1, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed Target Transcript Amplification and Sequencing (TATA-seq) to profile RNA within membrane-less organelles. This method enables sensitive and accurate study of RNA dynamics in cellular structures like stress granules.

Keywords:
in situ RNA reverse transcriptionlinear amplificationstress granulesubcellular localization

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Genomics

Background:

  • Membrane-less organelles, including P-bodies, stress granules, and nuclear speckles, are crucial for RNA regulation.
  • These dynamic structures form via liquid-liquid phase separation (LLPS) of RNA-binding proteins (RBPs) and RNA.
  • Current methods for studying RNA within these organelles lack sensitivity, specificity, and simplicity.

Purpose of the Study:

  • To develop a novel, sensitive, and specific method for profiling RNA within membrane-less organelles.
  • To overcome limitations of existing techniques for RNA analysis in subcellular compartments.

Main Methods:

  • Introduced Target Transcript Amplification and Sequencing (TATA-seq), a technique for in situ RNA profiling.
  • Utilized antibody-based targeting of organelle marker proteins to recruit a T7 promoter.
  • Employed in situ reverse transcription and T7 RNA polymerase amplification for sequencing library preparation.
  • Incorporated IgG controls for background noise subtraction.

Main Results:

  • Successfully profiled RNA in stress granules induced by sodium arsenite in HeLa cells.
  • Achieved high mapping ratios (approx. 90%) and controlled duplication rates (≤25%).
  • Validated TATA-seq findings using Fluorescence In Situ Hybridization (FISH) and immunofluorescence colocalization.

Conclusions:

  • TATA-seq provides a simple, sensitive, and accurate approach for studying RNA dynamics in membrane-less organelles.
  • This method significantly advances the capabilities for investigating RNA biology in subcellular structures.
  • TATA-seq facilitates a deeper understanding of RNA's role in cellular processes regulated by membrane-less organelles.