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Analyzing RNA Localization Using the RNA Proximity Labeling Method OINC-seq.

Megan C Pockalny¹, Hei-Yong G Lo^1,2, Raeann Goering^1,2

¹Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.

|August 13, 2025

Summary

This summary is machine-generated.

We developed OINC-seq, a novel method to map RNA localization within cells. This technique uses proximity-dependent RNA oxidation and sequencing to identify the RNA content of specific subcellular locations.

Keywords:

Proximity labeling RNA localization RNA modifications RNA oxidation RNA trafficking

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Area of Science:

Molecular Biology
Cell Biology
Genomics

Background:

RNA localization is crucial for cell function, but the sequences directing this transport and the RNA content of subcellular locations are largely unknown.
Existing methods lack the resolution to comprehensively map subcellular transcriptomes.

Purpose of the Study:

To develop and validate a novel method, OINC-seq, for assaying RNA localization and characterizing subcellular transcriptomes.
To enable the study of RNA transport mechanisms and the functional roles of localized RNAs.

Main Methods:

OINC-seq employs photoactivatable, spatially restricted RNA oxidation to label RNAs near a bait protein.
Oxidative RNA marks are detected via high-throughput sequencing by observing reverse transcriptase misincorporation events.
The PIGPEN software package quantifies mutation rates to infer RNA proximity to the bait protein.

Main Results:

OINC-seq successfully assays the RNA content of diverse subcellular locations.
The method provides a quantitative measure of RNA proximity to localized proteins.
High-throughput sequencing readout of oxidative RNA marks requires no prior enrichment.

Conclusions:

OINC-seq is a powerful tool for studying subcellular transcriptomes and RNA localization.
This method advances our understanding of RNA transport and function within cells.
The combination of OINC-seq and PIGPEN offers a comprehensive approach to RNA mapping.