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Related Experiment Video

Updated: Jun 10, 2025

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Published on: July 10, 2020

Mapping multiple RNA modifications simultaneously by proximity barcode sequencing.

Erdem Sendinc^1,2,3, Hua Yu⁴, Yu Hsien Hwang Fu⁴

¹Stem Cell Program, Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA 02115, USA.

Biorxiv : the Preprint Server for Biology

|October 17, 2024

Summary

This summary is machine-generated.

EpiPlex RNA modification profiling enables simultaneous mapping of multiple RNA marks, advancing epitranscriptome studies. This new method reveals how METTL3 and EIF4A3 influence N6-methyladenosine (m⁶A) levels and distribution.

Keywords:

EIF4A3 METTL3 RNA modification epitranscriptome inosine m6A

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Last Updated: Jun 10, 2025

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

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Published on: July 10, 2020

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Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Published on: August 4, 2016

Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

The epitranscriptome encompasses diverse RNA chemical modifications, with N6-methyladenosine (m⁶A) on mRNA crucial for posttranscriptional gene regulation.
Existing technologies allow transcriptome-wide mapping of single RNA modifications, but lack methods for simultaneous multi-modification analysis.
Simultaneous detection of multiple RNA modifications within a single sample is a significant unmet need in epitranscriptome research.

Purpose of the Study:

To develop a novel, convenient method for simultaneous mapping of multiple RNA modifications.
To enable relative quantification of RNA modification abundance at specific loci.
To investigate the impact of METTL3 and EIF4A3 on the epitranscriptome.

Main Methods:

EpiPlex RNA modification profiling, a bead-based proximity barcoding assay with sequencing readout.
Utilizes molecular recognition for multi-target RNA modification detection with low RNA input.
Relative quantification achieved by measuring signal intensity against spike-in controls.

Main Results:

EpiPlex successfully mapped multiple RNA modifications simultaneously.
Pharmacological inhibition of METTL3 decreased m⁶A levels, while EIF4A3 inhibition/knockdown increased m⁶A levels near exon junctions.
EIF4A3 manipulation confirmed its role in shaping the m⁶A landscape, without affecting inosine levels.

Conclusions:

EpiPlex provides a reliable and convenient platform for simultaneous mapping of multiple RNA modifications.
The method facilitates deeper understanding of epitranscriptome dynamics and regulation.
Findings elucidate the roles of METTL3 and EIF4A3 in regulating m⁶A modification patterns.