Purification of enzymatically active Xrn1 for removal of non-capped mRNAs from in vitro transcription reactions and evaluation of mRNA decapping status in vivo

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Karolina Drążkowska1,#, Rafał Tomecki2,3,#,*, and Agnieszka Tudek2,* 1Laboratory of Epitranscriptomics, Department of Environmental Microbiology and Biotechnology, Institute of Microbiology, Faculty of Biology, Biological and Chemical Research Centre, University of Warsaw, Poland 2Laboratory of RNA Processing and Decay, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland 3Institute of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, Poland #Karolina Drążkowska and Rafał Tomecki contributed equally to this work *Correspondence should be addressed to Agnieszka Tudek or Rafał Tomecki Abstract The cap is a 7-methylguanosine attached to the first messenger RNA (mRNA) nucleotide with a 5'-5' triphosphate bridge. This conserved eukaryotic modification confers stability to the transcripts and is essential for translation initiation. The specific mechanisms that govern transcript cytoplasmic longevity and translatability were always of substantial interest. Multiple works aimed at modeling mRNA decay mechanism, including the onset of decapping, which is the rate-limiting step of mRNA decay. Additionally, with the recent advances in RNA-based vaccines, the importance of efficient synthesis of fully functional mRNA has increased. Non-capped mRNAs arising during in vitro transcription are highly immunogenic, and multiple approaches were developed to reduce their levels. Efficient and low-cost methods for elimination of non-capped mRNAs in vitro are therefore essential to basic sciences and to pharmaceutical applications. Here, we present a protocol for heterologous expression and purification of catalytically active recombinant Xrn1 from Thermothelomyces (Myceliophthora) thermophilus (Tt_Xrn1). We also describe protocols needed to verify the enzyme quality, and provide an overview of its potential applications.

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