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Mol Cell. 2018 May 3;70(3):553-564.e9. doi: 10.1016/j.molcel.2018.03.014. Epub 2018 Apr 19.

CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD<sup/>.

Author information

1
Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
2
Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA; Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA.
3
Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA; Department of Biomedical and Health Informatics, Children's Hospital, Philadelphia, PA 19041, USA.
4
Key Laboratory of Synthetic Biology, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai 200032, China.
5
Department of Cell Biology and Neuroscience, Rutgers University, Piscataway, NJ 08854, USA.
6
Division of Biomolecular Physics, Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, 12116 Prague 2, Czech Republic.
7
Institute of Microbiology, Czech Academy of Sciences, v.v.i., Vídeňská 1083, 14220 Prague 4, Czech Republic.
8
Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA; Department of Biomedical and Health Informatics, Children's Hospital, Philadelphia, PA 19041, USA; Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Obstetrics, Gynecology and Reproductive Sciences, Rutgers Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA.
9
Department of Chemistry and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA. Electronic address: ebright@waksman.rutgers.edu.
10
Department of Genetics and Waksman Institute, Rutgers University, Piscataway, NJ 08854, USA. Electronic address: bnickels@waksman.rutgers.edu.

Abstract

Nucleoside-containing metabolites such as NAD+ can be incorporated as 5' caps on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report CapZyme-seq, a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-seq with multiplexed transcriptomics, we determine efficiencies of NAD+ capping by Escherichia coli RNAP for ∼16,000 promoter sequences. The results define preferred transcription start site (TSS) positions for NAD+ capping and define a consensus promoter sequence for NAD+ capping: HRRASWW (TSS underlined). By applying CapZyme-seq to E. coli total cellular RNA, we establish that sequence determinants for NCIN capping in vivo match the NAD+-capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs (sRNAs). Our findings define the promoter-sequence determinants for NCIN capping with NAD+ and provide a general method for analysis of NCIN capping in vitro and in vivo.

KEYWORDS:

NudC; RNA capping; RNA polymerase; RNA-seq; Rai1; nicotinamide adenine dinucleotide; non-canonical initiating nucleotide; transcription; transcription initiation; transcription start site

PMID:
29681497
PMCID:
PMC5935523
DOI:
10.1016/j.molcel.2018.03.014
[Indexed for MEDLINE]
Free PMC Article

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