mRNAs biotinylated within the 5′ cap and protected against decapping: new tools to capture RNA-protein complexes
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 synt...
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| Main Authors: | , , |
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| Format: | Article (Journal) |
| Language: | English |
| Published: |
05 November 2018
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| In: |
Philosophical transactions. Series B, Biological sciences
Year: 2018, Volume: 373, Issue: 1762 |
| ISSN: | 1471-2970 |
| DOI: | 10.1098/rstb.2018.0167 |
| Online Access: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1098/rstb.2018.0167 Verlag, lizenzpflichtig, Volltext: https://royalsocietypublishing.org/doi/10.1098/rstb.2018.0167 
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| Author Notes: | Sylwia Bednarek, Vanesa Madan, Pawel J. Sikorski, Ralf Bartenschlager, Joanna Kowalska and Jacek Jemielity |
| Summary: | 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’. |
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| Item Description: | Gesehen am 10.03.2020 |
| Physical Description: | Online Resource |
| ISSN: | 1471-2970 |
| DOI: | 10.1098/rstb.2018.0167 |