CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5′ Capping with NAD+

Irina O. Vvedenskaya, Jeremy G. Bird, Yuanchao Zhang, Yu Zhang, Xinfu Jiao, Ivan Barvík, Libor Krásný, Megerditch Kiledjian, Deanne M. Taylor, Richard Ebright, Bryce Nickels

Research output: Contribution to journalArticle

14 Scopus citations

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. Vvedenskaya et al. report a high-throughput-sequencing-based technology that employs NAD+-decapping enzymes to detect and quantify NAD+-capped RNA. Analysis of NAD+ capping for ∼16,000 promoter sequences defines a consensus promoter sequence for NAD+ capping.

Original languageEnglish (US)
Pages (from-to)553-564.e9
JournalMolecular cell
Volume70
Issue number3
DOIs
Publication statusPublished - May 3 2018

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All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Keywords

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

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