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.

中文翻译：

---

用于评估 XRN1 介导的衰变的工程病毒 RNA 衰变中间体

RNA 合成和衰变都必须在细胞内平衡以实现正确的基因表达。此外，RNA 衰变的调制专门为细胞提供了一个机会，可以响应特定的刺激或线索快速重塑转录组。因此，了解 RNA 衰变对基因表达稳态的潜在机制至关重要。无细胞重建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，很难在这样的体外环境中检查 RNA 衰变的所有方面，部分原因是通过变性聚丙烯酰胺凝胶解析较大 RNA 的能力受到限制。因此，研究 RNA 衰变的体外系统依赖于更小、生物学相关性较低的 RNA 片段。在此处，我们提出了一种方法，通过包含工程化的 XRN1 抗性报告 RNA (xrRNA)，更自信地检查大 mRNA 大小转录本的 RNA 衰减参数。通过将 67 个核苷酸的 xrRNA 放置在任何具有可变大小或序列背景的体外转录 RNA 的 3' 末端附近，研究人员可以观察 xrRNA 的积累，作为外切核糖核酸酶介导的 5'-3' 衰变的读数。这种方法可能允许体外 RNA 衰变分析包括完整的生物学相关 mRNA/mRNP，扩展其效用并允许改进实验设计考虑以促进生物学相关结果。在任何具有可变大小或序列背景的体外转录 RNA 结束时，研究人员可以观察 xrRNA 的积累，作为外切核糖核酸酶介导的 5'-3' 衰变的读数。这种方法可能允许体外 RNA 衰变分析包括完整的生物学相关 mRNA/mRNP，扩展其效用并允许改进实验设计考虑以促进生物学相关结果。在任何具有可变大小或序列背景的体外转录 RNA 结束时，研究人员可以观察 xrRNA 的积累，作为外切核糖核酸酶介导的 5'-3' 衰变的读数。这种方法可能允许体外 RNA 衰变分析包括完整的生物学相关 mRNA/mRNP，扩展其效用并允许改进实验设计考虑以促进生物学相关结果。