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Updated: May 9, 2026
Rup (RNA-seq Usability Assessment Pipeline) - Quality Control for Bulk RNA-seq Experiments in Eukaryotes
Published on: November 7, 2025
Direct assessment of transcription fidelity by high-resolution RNA sequencing
Masahiko Imashimizu1, Taku Oshima, Lucyna Lubkowska
1Gene Regulation and Chromosome Biology Laboratory, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD 21702, USA and Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.
Abstract:
Cancerous and aging cells have long been thought to be impacted by transcription errors that cause genetic and epigenetic changes. Until now, a lack of methodology for directly assessing such errors hindered evaluation of their impact to the cells. We report a high-resolution Illumina RNA-seq method that can assess noncoded base substitutions in mRNA at 10(-4)-10(-5) per base frequencies in vitro and in vivo. Statistically reliable detection of changes in transcription fidelity through ∼10(3) nt DNA sites assures that the RNA-seq can analyze the fidelity in a large number of the sites where errors occur. A combination of the RNA-seq and biochemical analyses of the positions for the errors revealed two sequence-specific mechanisms that increase transcription fidelity by Escherichia coli RNA polymerase: (i) enhanced suppression of nucleotide misincorporation that improves selectivity for the cognate substrate, and (ii) increased backtracking of the RNA polymerase that decreases a chance of error propagation to the full-length transcript after misincorporation and provides an opportunity to proofread the error. This method is adoptable to a genome-wide assessment of transcription fidelity.
Insights
Researchers developed a new RNA-sequencing method to detect transcription errors in cells. This method reveals how RNA polymerase mechanisms improve transcription accuracy, impacting cancer and aging research.
Area of Science:
- Molecular Biology
- Genetics
Background:
- Transcription errors are implicated in cellular aging and cancer.
- Previous methods lacked the sensitivity to directly assess these errors in mRNA.
Purpose of the Study:
- To develop a high-resolution method for assessing transcription fidelity.
- To investigate mechanisms enhancing transcription accuracy by RNA polymerase.
Main Methods:
- Utilized high-resolution Illumina RNA-sequencing to detect non-coded base substitutions in mRNA.
- Employed biochemical analyses to pinpoint error locations and mechanisms.
- Validated the method for in vitro and in vivo applications.
Main Results:
- Developed a method capable of detecting transcription errors at frequencies of 10^-4 to 10^-5.
- Identified two sequence-specific mechanisms employed by E. coli RNA polymerase to enhance fidelity.
- Mechanism 1: Improved nucleotide misincorporation suppression.
- Mechanism 2: Increased RNA polymerase backtracking for error correction and proofreading.
Conclusions:
- The new RNA-sequencing method enables genome-wide assessment of transcription fidelity.
- Understanding transcription error mechanisms provides insights into cellular aging and cancer.
- The findings highlight the sophisticated error-correction capabilities of RNA polymerase.
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