Engineered viral RNA decay intermediates to assess XRN1-mediated decay.
Engineered viral RNA decay intermediates to assess XRN1-mediated decay.
复制标题
设计病毒 RNA 衰变中间体以评估 XRN1 介导的衰变。
DOI：
10.1016/j.ymeth.2018.11.019
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发表时间：
2019
期刊：
影响因子：
--
通讯作者：
Wilusz,Jeffrey
中科院分区：
文献类型：
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作者：
Russo,Joseph;Mundell,CaryT;Charley,PhillidaA;Wilusz,Carol;Wilusz,Jeffrey
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 anin vitrosetting due, in part, to limitations on the ability to resolve larger RNAs through denaturing polyacrylamide gels. Thus,in vitrosystems 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 anyin vitrotranscribed 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 allowin vitroRNA decay assays to include full biologically relevant mRNA/mRNPs, extending their utility and allow improved experimental design considerations to promote biologically relevant outcomes.
