Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes

• Verfasst von: Weber, Frederik [VerfasserIn]
• Verfasst von: Motzkus, Nikolas [VerfasserIn]
• Verfasst von: Brandl, Leona [VerfasserIn]
• Verfasst von: Möhler, Marvin [VerfasserIn]
• Verfasst von: Alempijevic, Andrijana [VerfasserIn]
• Verfasst von: Jäschke, Andres [VerfasserIn]
• Titel: Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes
• Verf.angabe: Frederik Weber, Nikolas Alexander Motzkus, Leona Brandl, Marvin Möhler, Andrijana Alempijevic and Andres Jäschke
• E-Jahr: 2024
• Jahr: 8 May 2024
• Umfang: 13 S.
• Illustrationen: Illustrationen
• Fussnoten: Gesehen am 07.03.2025
• Titel Quelle: Enthalten in: Nucleic acids research
• Ort Quelle: Oxford : Oxford Univ. Press, 1974
• Jahr Quelle: 2024
• Band/Heft Quelle: 52(2024), 10 vom: Juni, Seite 5438-5450
• ISSN Quelle: 1362-4962
• DOI: doi:10.1093/nar/gkae353
• Datenträger: Online-Ressource
• Sprache: eng
• Sach-SW: DNA-Directed RNA Polymerases
• Sach-SW: Endoribonucleases
• Sach-SW: Escherichia coli
• Sach-SW: Humans
• Sach-SW: RNA Caps
• Sach-SW: Uridine Diphosphate N-Acetylglucosamine
• Sach-SW: Viral Proteins
• K10plus-PPN: 1919303995

Abstract

In recent years, several noncanonical RNA caps derived from cofactors and metabolites have been identified. Purine-containing RNA caps have been extensively studied, with multiple decapping enzymes identified and efficient capture and sequencing protocols developed for nicotinamide adenine dinucleotide (NAD)-RNA, which allowed for a stepwise elucidation of capping functions. Despite being identified as an abundant noncanonical RNA-cap, UDP-sugar-capped RNA remains poorly understood, which is partly due to its complex in vitro preparation. Here, we describe a scalable synthesis of sugar-capped uridine-guanosine dinucleotides from readily available protected building blocks and their enzymatic conversion into several cell wall precursor-capped dinucleotides. We employed these capped dinucleotides in T7 RNA polymerase-catalyzed in vitro transcription reactions to efficiently generate RNAs capped with uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), its N-azidoacetyl derivative UDP-GlcNAz, and various cell wall precursors. We furthermore identified four enzymes capable of processing UDP-GlcNAc-capped RNA in vitro: MurA, MurB and MurC from Escherichia coli can sequentially modify the sugar-cap structure and were used to introduce a bioorthogonal, clickable moiety, and the human Nudix hydrolase Nudt5 was shown to efficiently decap UDP-GlcNAc-RNA. Our findings underscore the importance of efficient synthetic methods for capped model RNAs. Additionally, we provide useful enzymatic tools that could be utilized in the development and application of UDP-GlcNAc capture and sequencing protocols. Such protocols are essential for deepening our understanding of the widespread yet enigmatic GlcNAc modification of RNA and its physiological significance.