In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells
In Vitro Transcribed RNA-based Luciferase Reporter Assay to Study Translation Regulation in Poxvirus-infected Cells
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DOI：
10.3791/59626
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发表时间：
2019-05-01
影响因子：
1.2
通讯作者：
Yang, Zhilong
中科院分区：
文献类型：
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作者：
Dhungel, Pragyesh;Cantu, Fernando;Yang, Zhilong
病毒DNA复制后转录的每个痘病毒mRNA在5 '-UTR中具有进化上保守的、非模板化的5'-poly（A）前导序列。为了剖析痘病毒感染期间5 '-poly（A）前导序列在mRNA翻译中的作用，我们开发了体外转录的基于RNA的荧光素酶报告基因测定。该报告基因测定包括四个核心步骤：（1）PCR扩增用于体外转录的DNA模板;（2）使用T7 RNA聚合酶体外转录以产生mRNA;（3）转染以将体外转录的mRNA引入细胞;（4）检测荧光素酶活性作为翻译的指示剂。本文所述的基于RNA的荧光素酶报告基因测定避免了痘病毒感染细胞中质粒复制和质粒隐蔽转录的问题。该方案可用于确定在除痘病毒感染的细胞以外的系统中mRNA（包括5 '-UTR和3'-UTR）中顺式元件的翻译调节。此外，不同模式的翻译起始，如帽依赖性，帽非依赖性，重新启动，和内部启动可以使用这种方法进行研究。
Every poxvirus mRNA transcribed after viral DNA replication has an evolutionarily conserved, non-templated 5'-poly(A) leader in the 5'-UTR. To dissect the role of 5'-poly(A) leader in mRNA translation during poxvirus infection we developed an in vitro transcribed RNA-based luciferase reporter assay. This reporter assay comprises of four core steps: (1) PCR to amplify the DNA template for in vitro transcription; (2) in vitro transcription to generate mRNA using T7 RNA polymerase; (3) Transfection to introduce in vitro transcribed mRNA into cells; (4) Detection of luciferase activity as the indicator of translation. The RNA-based luciferase reporter assay described here circumvents issues of plasmid replication in poxvirus-infected cells and cryptic transcription from the plasmid. This protocol can be used to determine translation regulation by cis-elements in an mRNA including 5'-UTR and 3'-UTR in systems other than poxvirus-infected cells. Moreover, different modes of translation initiation like cap-dependent, cap-independent, re-initiation, and internal initiation can be investigated using this method.