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<p>Although we know a great deal about the subcellular locations of most proteins, our knowledge of where most RNAs localize within cells remains limited. As RNA subcellular localization determines the fate, function and regulation of both coding and noncoding RNAs, there has been substantial interest in developing new scalable approaches to study the location of RNAs in their native context. Furthermore, many locations, such as membrane-bound and membrane-less organelles, have traditionally been challenging to study owing to lack of suitable tools to interrogate their constituents. Here we describe a detailed protocol for APEX sequencing (APEX-seq) that yields transcriptome-wide information of the subcellular address of RNAs that can, in principle, be applied to any subcellular location, membrane or condensate. APEX-seq utilizes a genetically encoded engineered ascorbate peroxidase (APEX2) tagged to a specific protein that localizes it to a region of interest. In the presence of biotin-phenol and hydrogen peroxide, APEX2 catalyzes the biotinylation of RNAs in its vicinity, which can be purified using streptavidin beads and sequenced to reveal the RNA repertoire at that subcellular location. APEX-seq experiments can be carried out by laboratory personnel trained in molecular biology. The analysis of APEX-seq data requires familiarity with standard RNA sequencing workflows. With APEX2-expressing cell lines in hand, the entire procedure from labeling reaction to analysis can be completed in 1 week. We expect this proximity labeling approach to facilitate the unbiased discovery of RNAs localizing to different organelles and to generate hypotheses for the mechanisms and pathways involved in regulating these processes.</p>

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APEX-seq maps transcriptome-wide subcellular RNA localization in living cells

  • 期刊论文

  • 发表日期：

    2026年4月2日

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  • Surbhi Sharma,
  • Madeline E. Rasband,
  • Xuemei Wang,
  • Karen Tolentino,
  • Sharon S. Huang,
  • Furqan M. Fazal

摘要

Although we know a great deal about the subcellular locations of most proteins, our knowledge of where most RNAs localize within cells remains limited. As RNA subcellular localization determines the fate, function and regulation of both coding and noncoding RNAs, there has been substantial interest in developing new scalable approaches to study the location of RNAs in their native context. Furthermore, many locations, such as membrane-bound and membrane-less organelles, have traditionally been challenging to study owing to lack of suitable tools to interrogate their constituents. Here we describe a detailed protocol for APEX sequencing (APEX-seq) that yields transcriptome-wide information of the subcellular address of RNAs that can, in principle, be applied to any subcellular location, membrane or condensate. APEX-seq utilizes a genetically encoded engineered ascorbate peroxidase (APEX2) tagged to a specific protein that localizes it to a region of interest. In the presence of biotin-phenol and hydrogen peroxide, APEX2 catalyzes the biotinylation of RNAs in its vicinity, which can be purified using streptavidin beads and sequenced to reveal the RNA repertoire at that subcellular location. APEX-seq experiments can be carried out by laboratory personnel trained in molecular biology. The analysis of APEX-seq data requires familiarity with standard RNA sequencing workflows. With APEX2-expressing cell lines in hand, the entire procedure from labeling reaction to analysis can be completed in 1 week. We expect this proximity labeling approach to facilitate the unbiased discovery of RNAs localizing to different organelles and to generate hypotheses for the mechanisms and pathways involved in regulating these processes.