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Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease

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RNA Modifications

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2298))

Abstract

The post-transcriptional modification of tRNAs at the wobble position plays a critical role in proper mRNA decoding and efficient protein synthesis. In particular, certain wobble uridines in eukaryotes are converted to 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). The mcm5s2U modification modulates decoding during translation by increasing the stringency of the wobble uridine to base pair with its canonical nucleotide partner, thereby restricting decoding to its cognate codon. Here, we outline a technique to monitor wobble uridine status in mcm5s2U-containing tRNAs using the gamma-toxin endonuclease from the yeast Kluyveromyces lactis that naturally cleaves tRNAs containing the mcm5s2U modification. This technique is coupled to Northern blotting or reverse transcription-PCR to enable rapid and sensitive detection of changes in mcm5s2U modification state.

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Acknowledgments

We thank Stewart Shuman at Memorial Sloan Kettering for the gamma-toxin expression plasmid along with Jillian Ramos and Kejia Zhang for comments on the chapter. This work was supported by NSF CAREER Award 1552126 to D.F.

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  1. Department of Biology, Center for RNA Biology, University of Rochester, Rochester, NY, USA

    Jenna M. Lentini & Dragony Fu

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  1. Jenna M. Lentini
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Correspondence to Dragony Fu .

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  1. Department of Urology, University of California, San Francisco, San Francisco, CA, USA

    Mary McMahon

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Lentini, J.M., Fu, D. (2021). Monitoring the 5-Methoxycarbonylmethyl-2-Thiouridine (mcm5s2U) Modification Utilizing the Gamma-Toxin Endonuclease. In: McMahon, M. (eds) RNA Modifications. Methods in Molecular Biology, vol 2298. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1374-0_13

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  • DOI: https://doi.org/10.1007/978-1-0716-1374-0_13

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