In situ visualization of m6A sites in cellular mRNAs

Author:

Sheehan Charles J1,Marayati Bahjat Fadi1,Bhatia Janvi2,Meyer Kate D13ORCID

Affiliation:

1. Department of Biochemistry, Duke University School of Medicine , Durham , NC , USA

2. Trinity College of Arts and Sciences, Duke University , Durham , NC , USA

3. Department of Neurobiology, Duke University School of Medicine , Durham , NC , USA

Abstract

Abstract N 6-methyladenosine (m6A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m6A is spatially regulated remains limited due to a lack of methods for visualizing methylated transcripts of interest in cells. Here, we develop DART-FISH, a method for in situ visualization of specific m6A sites in target RNAs which enables simultaneous detection of both m6A-modified and unmodified transcript copies. We demonstrate the ability of DART-FISH to visualize m6A in a variety of mRNAs across diverse cell types and to provide information on the location and stoichiometry of m6A sites at single-cell resolution. Finally, we use DART-FISH to reveal that m6A is not sufficient for mRNA localization to stress granules during oxidative stress. This technique provides a powerful tool for examining m6A-modified transcript dynamics and investigating methylated RNA localization in individual cells.

Funder

National Institutes of Health

Rita Allen Foundation

Publisher

Oxford University Press (OUP)

Subject

Genetics

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