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The two-step purification method ViREn identifies a single NSUN6-mediated 5-methylcytosine modification promoting dengue virus RNA genome turnover

Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes, including RNA stability and translation. Although advances in analytical methods have improved the detection of RNA modifications, precise mapping at single-base resolution rema...

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Main Authors: Wu, Chia Ching (Author) , Naarmann-de Vries, Isabel S. (Author) , Hartmann, Jonas (Author) , Nidoieva, Zarina (Author) , Kopietz, Kevin (Author) , Marchand, Virginie (Author) , Özrendeci, Zeynep (Author) , Lindner, Doris (Author) , Schelchshorn, Sophia (Author) , Flad, Sophia (Author) , Frye, Michaela (Author) , Papavasiliou, Nina (Author) , Schirmeister, Tanja (Author) , Stoecklin, Georg (Author) , Schott, Johanna (Author) , Motorin, Yuri (Author) , Tuorto, Francesca (Author) , Dieterich, Christoph (Author) , Helm, Mark (Author) , Ruggieri, Alessia (Author)
Format: Article (Journal)
Language:English
Published: 27 January 2026
In: Nucleic acids research
Year: 2026, Volume: 54, Issue: 2, Pages: 1-19
ISSN:1362-4962
DOI:10.1093/nar/gkag003
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.1093/nar/gkag003
Verlag, kostenfrei, Volltext: https://academic.oup.com/nar/article/54/2/gkag003/8433881
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Author Notes:Chia Ching Wu, Isabel S Naarmann-de Vries, Jonas Hartmann, Zarina Nidoieva, Kevin Kopietz, Virginie Marchand, Zeynep Özrendeci, Doris Lindner, Sophia Schelchshorn, Sophia Flad, Michaela Frye, F Nina Papavasiliou, Tanja Schirmeister, Georg Stoecklin, Johanna Schott, Yuri Motorin, Francesca Tuorto, Christoph Dieterich, Mark Helm, Alessia Ruggieri
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Summary:Chemical modifications on cellular and viral RNAs are new layers of post-transcriptional regulation of cellular processes, including RNA stability and translation. Although advances in analytical methods have improved the detection of RNA modifications, precise mapping at single-base resolution remains challenging. Requirements for sensitivity and purity limit accuracy and reproducibility, especially for low abundant viral RNAs extracted from infected cells. Here, we report a two-step method, ViREn, for the enrichment of the genomic RNA (gRNA) of dengue virus (DENV), a positive-sense single-stranded RNA virus. This approach enabled the preparation of gRNA with significantly increased purity and led to the identification of a single high-confidence 5-methylcytosine (m5C) site in DENV gRNA at position 1218. This finding was orthogonally validated by Illumina-based bisulfite sequencing and by Nanopore Oxford Technologies direct RNA sequencing. Strikingly, m5C1218 was detected exclusively in gRNA extracted from infected cells but not in gRNA extracted from viral particles. We identified NSUN6 as the host methyltransferase catalyzing this modification and demonstrated a role for m5C in regulating DENV gRNA turnover. ViREn thus enables the mapping of m5C on low-abundance viral gRNA with unprecedented precision and sensitivity and facilitates future mechanistic studies into the role of RNA modifications in virus replication.
Item Description:Online veröffentlicht: 21. Januar 2026
Gesehen am 10.02.2026
Physical Description:Online Resource
ISSN:1362-4962
DOI:10.1093/nar/gkag003

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