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Profiling m6A RNA Modifications in Low Amounts of Plant Cells Using Maize Meiocytes
Drin Shabani1, Thomas Dresselhaus1, Stefanie Dukowic-Schulze2
1Department of Cell Biology and Plant Biochemistry, University of Regensburg, Regensburg, Germany.
Abstract:
RNA modifications can influence gene expression via multiple aspects such as RNA stability and alternative splicing. The most prominent RNA modification is m6A (N6-methyladenosine). Its profiling from low starting amounts of <100 cells is challenging. We describe here a complete workflow from cell isolation to data analysis that is based on using an RNA CUT&RUN-supported m6A-RIP (RNA immunoprecipitation) procedure and a subsequent adaptor-tagging library synthesis. Male meiocytes isolated from maize anthers were used as a test system to establish the protocol.
Insights
Profiling N6-methyladenosine (m6A) RNA modifications in less than 100 cells is difficult. This study presents a new workflow using RNA CUT&RUN-supported m6A-RNA immunoprecipitation (RIP) for efficient m6A profiling from limited cell samples.
Area of Science:
- Molecular Biology
- Epigenetics
- Plant Science
Background:
- RNA modifications regulate gene expression through stability and splicing.
- N6-methyladenosine (m6A) is a prominent RNA modification.
- Profiling m6A in low cell numbers (<100) presents significant technical challenges.
Purpose of the Study:
- To develop and validate a robust workflow for m6A profiling from limited cellular material.
- To establish a method for analyzing m6A modification patterns in specific cell types.
Main Methods:
- Development of an RNA CUT&RUN-supported m6A-RNA immunoprecipitation (RIP) protocol.
- Adaptation of adaptor-tagging library synthesis for high-throughput sequencing.
- Establishment of a complete workflow from cell isolation to data analysis.
Main Results:
- Successfully established a complete workflow for m6A profiling from low cell inputs.
- Demonstrated the feasibility of the protocol using male meiocytes from maize anthers.
- The workflow enables sensitive detection and analysis of m6A modifications.
Conclusions:
- The described RNA CUT&RUN-supported m6A-RIP workflow overcomes limitations in profiling m6A from scarce biological samples.
- This method provides a valuable tool for studying m6A epitranscriptomics in various biological contexts, including plant reproductive cells.
