mRNAs biotinylated within the 5′ cap and protected against decapping

• Verfasst von: Bednarek, Sylwia [VerfasserIn]
• Verfasst von: Madan Renes, Vanesa [VerfasserIn]
• Verfasst von: Bartenschlager, Ralf [VerfasserIn]
• Titel: mRNAs biotinylated within the 5′ cap and protected against decapping
• Titelzusatz: new tools to capture RNA-protein complexes
• Verf.angabe: Sylwia Bednarek, Vanesa Madan, Pawel J. Sikorski, Ralf Bartenschlager, Joanna Kowalska and Jacek Jemielity
• E-Jahr: 2018
• Jahr: 05 November 2018
• Umfang: 12 S.
• Fussnoten: Gesehen am 10.03.2020
• Titel Quelle: Enthalten in: Philosophical transactions / B
• Ort Quelle: London : Royal Society, 1990
• Jahr Quelle: 2018
• Band/Heft Quelle: 373(2018,1762) Artikel-Nummer 20180167, 12 Seiten
• ISSN Quelle: 1471-2970
• DOI: doi:10.1098/rstb.2018.0167
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1691985651

Abstract

The 5′-terminus of eukaryotic mRNAs comprises a 7-methylguanosine cap linked to the first transcribed nucleotide via a 5′-5′ triphosphate bond. This cap structure facilitates numerous interactions with molecules participating in mRNA processing, turnover and RNA translation. Here, we report the synthesis and biochemical properties of a set of biotin-labelled cap analogues modified within the triphosphate bridge and increasing mRNA stability while retaining biological activity. Successful co-transcriptional incorporation of the cap analogues allowed for the quantification of cap-dependent translation efficiency, capping efficiency and the susceptibility to decapping by Dcp2. The utility of such cap-biotinylated RNAs as molecular tool was demonstrated by ultraviolet-cross-linking and affinity capture of protein-RNA complexes. In conclusion, RNAs labelled with biotin via the 5′ cap structure can be applied to a variety of biological experiments based on biotin-avidin interaction or by means of biotin-specific antibodies, including protein affinity purification, pull-down assays, in vivo visualization, cellular delivery and many others.This article is part of the theme issue ‘5′ and 3′ modifications controlling RNA degradation’.