Journal
JOURNAL OF MOLECULAR BIOLOGY
Volume 432, Issue 13, Pages 3933-3949Publisher
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2020.04.011
Keywords
transcription fidelity; RNA polymerase I; Ribosomal RNA; RNA sequencing
Categories
Funding
- Shenzhen Science and Technology Innovation Committee [JCYJ20170413173837121]
- Innovation and Technology Commission [ITCPD/17-9]
- Hong Kong Research Grant Council [HKUST C6009-15G, AoE/P-705/16, T13-605/18-W]
- National Institute of Health [GM25232]
- NSFC [31922088]
- Hong Kong ITC [ITCPD/17-9, ITS/480/18FP]
- Hong Kong RGC [26102719, N_ HKUST606/17, C6002-17GF, C7065-18GF, R4017-18]
- Institute of Advanced Studies postdoctoral fellowship
- GRF [16301319]
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RNA polymerase transcribes certain genomic loci with higher errors rates. These transcription error-enriched genomic loci (TEELs) have implications in disease. Current deep-sequencing methods cannot distinguish TEELs from post-transcriptional modifications, stochastic transcription errors, and technical noise, impeding efforts to elucidate the mechanisms linking TEELs to disease. Here, we describe background error model-coupled precision nuclear run-on circular-sequencing (EmPC-seq) to discern genomic regions enriched for transcription misincorporations. EmPC-seq innovatively combines a nuclear run-on assay for capturing nascent RNA before post-transcriptional modifications, a circular-sequencing step that sequences the same nascent RNA molecules multiple times to improve accuracy, and a statistical model for distinguishing error-enriched regions among stochastic polymerase errors. Applying EmPC-seq to the ribosomal RNA transcriptome, we show that TEELs of RNA polymerase I are not randomly distributed but clustered together, with higher error frequencies at nascent transcript 3' ends. Our study establishes a reliable method of identifying TEELs with nucleotide precision, which can help elucidate their molecular origins. (C) 2020 Elsevier Ltd. All rights reserved.
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