m⁶A RNA modifications are measured at single-base resolution across the mammalian transcriptome

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• Functional studies of RNA N⁶-methyladenosine (m⁶A) modifications are hindered by the lack of methods to map individual m⁶A sites genome-wide.
• Existing techniques have limitations in resolution, quantification, or RNA input requirements.

Purpose of the Study:

• To introduce a novel method, m⁶A-selective allyl chemical labeling and sequencing (m⁶A-SAC-seq), for quantitative, whole-transcriptome mapping of m⁶A at single-nucleotide resolution.
• To enable detailed functional studies of m⁶A modification dynamics in biological processes.

Main Methods:

• Development and application of m⁶A-SAC-seq, a chemical labeling and sequencing approach.
• Requires minimal RNA input (~30 ng) from poly(A) or rRNA-depleted RNA.
• Mapping of m⁶A modification stoichiometries in cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs).

Main Results:

• Successful quantitative, whole-transcriptome mapping of m⁶A sites at single-nucleotide resolution.
• Identification of numerous cell-state-specific m⁶A sites with dynamic methylation status during cell differentiation.
• Observation of changes in m⁶A stoichiometry correlating with transcript expression of key transcriptional factors during HSPC differentiation.

Conclusions:

• m⁶A-SAC-seq is a powerful, quantitative tool for dissecting the dynamics and functional roles of m⁶A modifications.
• The method is applicable to diverse biological processes and requires limited RNA input.
• Provides new insights into the regulatory roles of m⁶A during cell differentiation and gene expression.