Direct assessment of transcription fidelity by high-resolution RNA sequencing.
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.
Gene 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.
Masahiko
M
Imashimizu
Taku
T
Oshima
Lucyna
L
Lubkowska
Mikhail
M
Kashlev
D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods*; D014158:Transcription, Genetic
D012321:DNA-Directed RNA Polymerases