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

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Nucleoside metabolites like NAD+ can function as non-canonical initiating nucleotides (NCINs) during RNA transcription.
• RNA polymerase (RNAP) can incorporate these NCINs as 5' caps on RNA molecules.
• Understanding the mechanisms and sequence determinants of NCIN capping is crucial for RNA biology.

Purpose of the Study:

• To develop a high-throughput sequencing method for detecting and quantifying NCIN-capped RNA.
• To determine the promoter sequence determinants for NAD+ capping by Escherichia coli RNAP.
• To investigate the in vivo relevance of identified capping sequences in E. coli.

Main Methods:

• Developed CapZyme-seq, a method utilizing NCIN-decapping enzymes NudC and Rai1.
• Combined CapZyme-seq with multiplexed transcriptomics to analyze ~16,000 promoter sequences.
• Applied CapZyme-seq to total cellular RNA from E. coli to study in vivo capping.

Main Results:

• Identified preferred transcription start site (TSS) positions for NAD+ capping.
• Defined a consensus promoter sequence (HRRASWW) for NAD+ capping by E. coli RNAP.
• Demonstrated that in vivo sequence determinants for NCIN capping match in vitro findings and identified NCIN-capped small RNAs (sRNAs).

Conclusions:

• Established promoter sequence determinants for NCIN capping with NAD+.
• Provided a versatile CapZyme-seq method for analyzing NCIN capping in vitro and in vivo.
• Advanced understanding of RNA capping mechanisms and their regulation by promoter sequences.