Nuclear-m⁶A-Label-Seq Enables Transcriptome-Wide Nuclear m⁶A Profiling at Single-Base Resolution.

Area of Science:

• Molecular Biology
• Epigenetics
• RNA Biology

Background:

• N⁶-Methyladenosine (m⁶A) modification is crucial in mRNA regulation, but its role in nuclear noncoding RNAs is less understood.
• Existing methods for m⁶A profiling lack single-base resolution or are not optimized for nuclear RNA.

Purpose of the Study:

• To develop and validate a novel method, Nuclear-m⁶A-label-seq, for high-resolution, transcriptome-wide mapping of m⁶A in nuclear noncoding RNAs.
• To provide a detailed protocol for efficient nuclear m⁶A epitranscriptome profiling.

Main Methods:

• Nuclear-m⁶A-label-seq utilizes metabolic labeling with an allyl group at adenosine sites, followed by conversion to cyclized adenosine (cyc-A).
• HIV reverse transcriptase induces base misincorporation at cyc-A sites during cDNA synthesis, enabling single-base resolution mapping.
• The protocol includes sequential nuclear RNA isolation, rRNA depletion, and analysis using the rMATS-DVR pipeline.

Main Results:

• Nuclear-m⁶A-label-seq significantly reduces RNA input (5 μg) and library construction time (~6 h) compared to previous methods.
• The method captures both polyadenylated and nonpolyadenylated nuclear transcripts.
• Accurate mapping of m⁶A sites, including in repetitive regions, is achieved by aligning reads to the T2T-CHM13 human genome.

Conclusions:

• Nuclear-m⁶A-label-seq is a direct, high-resolution, and efficient approach for profiling the nuclear m⁶A epitranscriptome.
• This method facilitates the study of m⁶A functions in nuclear noncoding RNAs.
• The detailed protocol enables broader adoption and advancement in epitranscriptomic research.