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Defining expansions and perturbations to the RNA polymerase III transcriptome and epitranscriptome by modified direct
Ruth Verstraten1,2, Pierina Cetraro1, Amy H Fitzpatrick1
1Institute of Virology, Hannover Medical School, Hannover, Germany.
Biorxiv : the Preprint Server for Biology
|March 31, 2025
Summary
A new method called DRAP3R (Direct Read and Analysis of Polymerase III transcribed RNAs) precisely analyzes RNA polymerase III (Pol III) transcribed RNAs and their modifications, revealing new RNA genes and modifications.
Area of Science:
- Molecular Biology
- Genomics
- RNA Biology
Background:
- RNA polymerase III (Pol III) transcribes essential non-coding RNAs, but its transcription and RNA modifications are poorly understood.
- Existing methods lack sensitivity and systematic analysis for Pol III transcribed RNAs.
Purpose of the Study:
- To develop a sensitive and systematic method for analyzing nascent Pol III transcribed RNAs and their modifications.
- To expand the understanding of the Pol III transcriptome and epitranscriptome.
Main Methods:
- Developed DRAP3R (Direct Read and Analysis of Polymerase III transcribed RNAs), a modified nanopore direct RNA sequencing approach.
- Applied DRAP3R to various cell types, including those infected with Herpes Simplex Virus Type 1.
Main Results:
- Identified previously unconfirmed tRNA genes and novel Pol III transcribed RNAs, expanding the known Pol III transcriptome.
- Enabled single-nucleotide resolution discrimination of RNA modifications like pseudouridine (Ψ) and N6-methyladenosine (m6A).
- Revealed differential Ψ installation patterns and extensive Pol III transcriptome/epitranscriptome remodeling upon viral infection.
Conclusions:
- DRAP3R is a powerful tool for systematic analysis of Pol III transcribed RNAs and their modifications.
- Provides new insights into the Pol III transcriptome, epitranscriptome, and their regulation in cellular processes and viral infections.
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