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A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
Published on: December 5, 2016
Detection of m6A RNA modifications at single-nucleotide resolution using m6A-selective allyl chemical labeling and
Yong Peng1, Hanzhe Meng2, Ruiqi Ge3
1Department of Chemistry, Institute for Biophysical Dynamics, Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA; Section of Genetic Medicine, Department of Medicine, Department of Human Genetics, The University of Chicago, Chicago, IL, USA.
Abstract:
As the most abundant internal mRNA modification, N6-methyladenosine (m6A) was involved in almost all the aspects of RNA metabolism. Here, we introduce our protocol for m6A-SAC-seq, which enables the whole transcriptome-wide mapping of m6A RNA modification at single-nucleotide resolution with stoichiometry information. m6A-SAC-seq relies on selective allyl labeling of m6A by specific methyltransferase and chemical treatment that introduce mutation upon reverse transcription. The technique only requires ∼30 ng of input RNA. For complete details on the use and execution of this protocol, please refer to Hu et al. (2022).
Insights
This study presents m6A-SAC-seq, a new method for mapping N6-methyladenosine (m6A) RNA modifications across the entire transcriptome. The technique provides single-nucleotide resolution and stoichiometry information with minimal RNA input.
Area of Science:
- Molecular Biology
- Epigenetics
- RNA Biology
Background:
- N6-methyladenosine (m6A) is the most prevalent internal modification in eukaryotic messenger RNA (mRNA).
- m6A modification plays a crucial role in regulating diverse RNA metabolic processes.
- Understanding the precise distribution of m6A is vital for deciphering its functional significance.
Purpose of the Study:
- To introduce and validate a novel protocol, m6A-SAC-seq, for high-resolution transcriptome-wide mapping of m6A.
- To enable the simultaneous assessment of m6A modification stoichiometry.
- To provide a sensitive method requiring minimal RNA input.
Main Methods:
- m6A-SAC-seq utilizes selective allyl labeling of m6A residues mediated by a specific methyltransferase.
- A subsequent chemical treatment introduces mutations during reverse transcription, enabling detection.
- The protocol is designed for whole transcriptome analysis at single-nucleotide resolution.
Main Results:
- m6A-SAC-seq successfully maps m6A modifications across the transcriptome.
- The method achieves single-nucleotide resolution, pinpointing modification sites accurately.
- Stoichiometry information regarding m6A modification levels can be obtained.
Conclusions:
- m6A-SAC-seq is an efficient and sensitive technique for mapping m6A RNA modifications.
- The protocol requires a low input amount of RNA (approximately 30 ng).
- This method facilitates comprehensive studies of m6A regulatory mechanisms and functions.
