Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States.
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80525, United States; Program in Cell & Molecular Biology, Colorado State University, Fort Collins, CO 80525, United States.
Methods. 2019 Feb 15;155:116-123. doi: 10.1016/j.ymeth.2018.11.019. Epub 2018 Dec 3.
Both RNA synthesis and decay must be balanced within a cell to achieve proper gene expression. Additionally, modulation of RNA decay specifically offers the cell an opportunity to rapidly reshape the transcriptome in response to specific stimuli or cues. Therefore, it is critical to understand the underlying mechanisms through which RNA decay contribute to gene expression homeostasis. Cell-free reconstitution approaches have been used successfully to reveal mechanisms associated with numerous post-transcriptional RNA processes. Historically, it has been difficult to examine all aspects of RNA decay in such an in vitro setting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus, in vitro systems to study RNA decay rely on smaller, less biologically relevant RNA fragments. Herein, we present an approach to more confidently examine RNA decay parameters of large mRNA size transcripts through the inclusion of an engineered XRN1-resistant reporter RNA (xrRNA). By placing a 67 nucleotide xrRNA near the 3' end of any in vitro transcribed RNA with variable size or sequence context, investigators can observe the accumulation of the xrRNA as a readout of exoribonuclease-mediated 5'-3' decay. This approach may allow in vitro RNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.
在细胞内，必须平衡 RNA 的合成和降解，以实现适当的基因表达。此外，RNA 降解的调节特别为细胞提供了一个机会，可以快速响应特定刺激或线索重塑转录组。因此，了解 RNA 降解对基因表达稳态的贡献的潜在机制至关重要。无细胞重构方法已成功用于揭示与许多转录后 RNA 过程相关的机制。从历史上看，由于在变性聚丙烯酰胺凝胶中解析较大 RNA 的能力有限，因此很难在这种体外环境中检查 RNA 降解的所有方面。因此，用于研究 RNA 降解的体外系统依赖于较小的、不太具有生物学相关性的 RNA 片段。在此，我们通过引入工程化的 XRN1 抗性报告 RNA（xrRNA），提出了一种更有信心地检查大 mRNA 大小转录物 RNA 降解参数的方法。通过将 67 个核苷酸的 xrRNA 放置在任何体外转录的 RNA 的 3' 端附近，该 RNA 的大小或序列上下文可变，研究人员可以观察 xrRNA 的积累，作为外切核酸酶介导的 5'-3' 降解的读数。这种方法可以使体外 RNA 降解测定法包括完整的具有生物学相关性的 mRNA/mRNP，从而扩展它们的用途，并允许改进实验设计考虑因素以促进具有生物学相关性的结果。
