Identification and in vitro characterization of UDP-GlcNAc-RNA cap-modifying and decapping enzymes
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 dinucleot...
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| Main Authors: | , , , , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
8 May 2024
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| In: |
Nucleic acids research
Year: 2024, Volume: 52, Issue: 10, Pages: 5438-5450 |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/gkae353 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1093/nar/gkae353
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| Author Notes: | Frederik Weber, Nikolas Alexander Motzkus, Leona Brandl, Marvin Möhler, Andrijana Alempijevic and Andres Jäschke |
| Summary: | 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. |
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| Item Description: | Gesehen am 07.03.2025 |
| Physical Description: | Online Resource |
| ISSN: | 1362-4962 |
| DOI: | 10.1093/nar/gkae353 |