CapZyme-Seq Comprehensively Defines Promoter-Sequence Determinants for RNA 5' Capping with NAD.
Mol Cell
; 70(3): 553-564.e9, 2018 05 03.
Article
em En
| MEDLINE
| ID: mdl-29681497
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 â¼16,000 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Capuzes de RNA
/
Regiões Promotoras Genéticas
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Sequenciamento de Nucleotídeos em Larga Escala
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NAD
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Mol Cell
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Estados Unidos