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.
Nucleic Acids Res. 2013 Oct;41(19):9090-104. doi: 10.1093/nar/gkt698. Epub 2013 Aug 7.
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.
长期以来，人们一直认为癌变和衰老细胞受到转录错误的影响，这些错误会导致遗传和表观遗传变化。直到现在，缺乏直接评估这些错误的方法学手段，阻碍了对其对细胞影响的评估。我们报告了一种高分辨率的 Illumina RNA-seq 方法，该方法可以在体外和体内以每碱基 10(-4)-10(-5)的频率评估 mRNA 中非编码碱基替换。通过大约 10(3)个 nt DNA 位点对转录保真度的统计上可靠的检测，确保了 RNA-seq 可以分析大量发生错误的位点的保真度。RNA-seq 与对错误位置的生化分析相结合，揭示了大肠杆菌 RNA 聚合酶提高转录保真度的两种序列特异性机制：（i）增强了对核苷酸错误掺入的抑制作用，从而提高了对同源底物的选择性；（ii）RNA 聚合酶的回溯增加，减少了错误掺入后全长转录物错误传播的机会，并提供了一个校对错误的机会。该方法可用于全基因组转录保真度评估。
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