Wang Yuqing, Chong Tin Hang, Unarta Ilona Christy, Xu Xinzhou, Suarez Gianmarco D, Wang Jiguang, Lis John T, Huang Xuhui, Cheung Peter Pak-Hang
The Hong Kong University of Science and Technology -Shenzhen Research Institute, Shenzhen, China.
Bioengineering Graduate Program, Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Hong Kong SAR.
Bio Protoc. 2021 Feb 20;11(4):e3921. doi: 10.21769/BioProtoc.3921.
Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer's. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be used to identify transcriptional mutations. Here we present background Error Model-coupled Precision nuclear run-on Circular-sequencing (EmPC-seq), a method combining a nuclear run-on assay and circular sequencing with a background error model to precisely detect nascent transcription errors and effectively discern TEELs within the genome.
转录错误可通过改变表观基因组并导致mRNA错配掺入，进而显著影响生物体的代谢过程，而这些错配的mRNA会被翻译成异常的突变蛋白。此外，在真核生物基因组中存在特定的富含转录错误的基因组位点（TEELs），这些位点由错误率显著更高的RNA聚合酶转录，据推测与癌症、衰老以及唐氏综合征和阿尔茨海默病等疾病有关。因此，转录错误的研究在遗传学领域日益重要。然而，方法学上的障碍限制了准确识别转录错误方面的进展。Pro-Seq和NET-Seq可以纯化新生RNA并沿基因组定位RNA聚合酶，但不能用于识别转录突变。在此，我们介绍背景误差模型耦合的精确核延伸循环测序（EmPC-seq），这是一种将核延伸分析和循环测序与背景误差模型相结合的方法，用于精确检测新生转录错误并有效识别基因组中的TEELs。
