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

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.

卡罗利娜·德拉日科夫斯卡¹, #, 拉法尔·托梅茨基²,³, #, *, 以及阿格涅什卡·图德克², *

¹ 波兰华大学生物与化学研究中心生物学院微生物研究所环境微生物学与生物技术系表观转录组学实验室
² 波兰科学院生物化学与生物物理研究所RNA加工与降解实验室，华沙
³ 波兰华大学生物学院遗传学与生物技术研究所

# 卡罗利娜·德拉日科夫斯卡与拉法尔·托梅茨基对本研究贡献均等
* 通讯作者可联系阿格涅什卡·图德克或拉法尔·托梅茨基

摘要
帽结构是一类以5'-5'三磷酸桥连接至首段信使RNA（messenger RNA, mRNA）核苷酸的7-甲基鸟苷（7-methylguanosine）修饰。这一保守的真核生物修饰可赋予转录本稳定性，并对翻译起始至关重要。长期以来，调控转录本细胞质半衰期与翻译效率的特异性机制均为研究热点。已有多项研究致力于构建mRNA降解机制模型，其中包括作为mRNA降解限速步骤的脱帽反应（decapping）的启动过程。此外，随着近年来基于RNA的疫苗研发进展，高效合成具备完全功能的mRNA的重要性日益凸显。体外转录过程中产生的未加帽mRNA具有高度免疫原性，目前已开发出多种方法以降低其含量。因此，能够高效且低成本地体外去除未加帽mRNA的方法，对基础科学研究与制药应用均至关重要。本文报道了一套用于异源表达与纯化来自嗜热热囊霉（Thermothelomyces (Myceliophthora) thermophilus）的具有催化活性的重组Xrn1（Tt_Xrn1）的实验方案。同时，本文还描述了验证该酶质量的相关实验流程，并概述了其潜在应用场景。