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Updated: Apr 22, 2026
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
Published on: February 18, 2022
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Harnessing toxin-mediated ribosome stalling as a complementary tool to annotate bacterial ORFs
Eduardo A Troian1, Valdir C Barth1,2, Unnati Chauhan1
1Department of Biochemistry and Molecular Biology, Rutgers University, Robert Wood Johnson Medical School, Piscataway, NJ 08854, United States.
Nucleic Acids Research
|April 21, 2026
Summary
Mycobacterium tuberculosis toxin VapC4 causes ribosome stalling, revealing new Cys-containing open reading frames (ORFs). This novel method, using ribosome stalling, efficiently detects and validates previously unannotated ORFs in Mtb genomes.
Area of Science:
- Microbiology
- Genomics
- Molecular Biology
Background:
- The Mycobacterium tuberculosis (Mtb) VapC4 toxin targets and inactivates tRNACys, causing ribosome stalling at cysteine codons.
- Existing methods for identifying novel open reading frames (ORFs) can be limited.
Purpose of the Study:
- To develop and validate a novel genome annotation tool using toxin-induced ribosome stalling.
- To identify and characterize previously unannotated cysteine-containing ORFs in Mtb.
Main Methods:
- Utilized 5' RNA-seq to map ribosome positions stalled by VapC4 toxin.
- Employed proteogenomics and mass spectrometry for ORF validation.
- Compared results with previously published Ribo-RET data.
Main Results:
- Successfully mapped stalled ribosomes to identify new Cys-containing Mtb ORFs without requiring Ribo-seq.
- Unmasked 96 unannotated ORFs, with 54% being small ORFs (≤50 amino acids).
- Validated 69% of the identified ORFs via mass spectrometry, including four matched to synthetic controls.
Conclusions:
- Toxin-mediated ribosome stalling is a robust and innovative genome annotation tool for mycobacteria and other bacteria.
- This method complements existing genome annotation techniques and offers unique advantages.
- Identified ORFs provide functional insights, including potential roles in Cys-responsiveness and encoding novel proteins.
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