“CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

Vvedenskaya, I. O.; Bird, J. G.; Zhang, Y.; Jiao, X.; Barvik, I.; Krasny, L.; Kiledjian, M.; Taylor, D. M.; Ebright, R. H.; Nickels, B. E.

doi:10.1101/239426

bioRxiv · 10.1101/239426

“CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+

Vvedenskaya, I. O.·Bird, J. G.·Zhang, Y.·Jiao, X.·Barvik, I.·Krasny, L.·Kiledjian, M.·Taylor, D. M.·Ebright, R. H.·Nickels, B. E.

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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. 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.

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Vvedenskaya, I. O., Bird, J. G., Zhang, Y., Jiao, X., Barvik, I., Krasny, L., Kiledjian, M., Taylor, D. M., Ebright, R. H., Nickels, B. E.. 2017-12-24. “CapZyme-Seq” comprehensively defines promoter-sequence determinants for RNA 5’ capping with NAD+. https://doi.org/10.1101/239426

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