体外转录 rna 基荧光素酶记者法研究类病毒感染细胞的翻译规律
Summary
我们提出了一种研究 mrna 翻译调节的方案, 在天花病毒感染细胞使用体外转录 Rna 基荧光素酶报告法。该方法可用于研究 mRNA 的cis元素的翻译调控, 包括 5 ‘-未翻译区域 (utr) 和 3 ‘-utr。使用此方法还可以检查不同的翻译启动模式。
Abstract
病毒 DNA 复制后转录的每一个痘病毒 mRNA 都有一个进化保守的、非模板化的 5 ‘-聚 (A) 引线在 5 ‘-utr 中。为了剖析 5 ‘-聚 (A) 引线在天花病毒感染过程中 mRNA 翻译中的作用, 我们开发了一种体外转录 rna 基荧光素酶报告法。本报告分析包括四个核心步骤: (1) PCR 扩增 DNA 模板进行体外转录;(2) 利用 T7 RNA 聚合酶进行体外转录生成 mRNA;(3) 转染, 将体外转录的 mRNA 引入细胞;(4) 检测荧光素酶活性作为翻译指标。本文介绍的基于 rna 的荧光素酶报告法规避了弓形虫感染细胞中质粒复制和质粒中的神秘转录问题。该协议可用于确定 mRNA 中的 cis元素在 poxvirus-infected 感染细胞以外的系统中的翻译调控, 包括 5 ‘-utr 和 3 ‘-utr。此外, 还可以利用这种方法研究不同的翻译启动模式, 如与上限相关、与上限无关、重新启动和内部启动。
Introduction
根据中心教条, 遗传信息从 dna 流向 rna, 然后最后流向蛋白质1,2。遗传信息的这种流动在许多层面上受到高度调控, 包括 mrna 翻译3,4。研究用记者检测来衡量基因表达的调控, 将有助于了解这一过程中涉及的调控机制。在这里, 我们描述了一种研究 mrna 翻译的方案, 使用体外转录 rna 为基础的荧光素酶报告法在 pxvirs 感染细胞。
痘病毒包括许多高度危险的人类和动物病原体5。与所有其他病毒一样, 弓形病毒完全依靠宿主细胞机制进行蛋白质合成6,7,8。为了有效地合成病毒蛋白, 病毒进化出许多策略来劫持细胞翻译机制, 将其重定向, 用于病毒 rna 7,8的翻译。病毒常用的一种机制是在其转录中使用cis作用元素。值得注意的例子包括内部核糖体进入网站 (ires) 和与上限无关的翻译增强剂 (cite)9、10、11。这些cis元素通过通过不同的机制吸引翻译机械 12,13, 14,使病毒转录具有平移优势。超过 100个 poxvirus mrna 在 5 ‘-未翻译区域 (5 ‘-utr) 中具有进化保守的cis作用元素: 在这些 mrna15,16的 5 ‘-端的 5 ‘-多边形 (a) 引线。这些 5 ‘ poly (a) 引线的长度是异质性的, 是由 poxvirus-c金编码 rna 聚合酶在转录17,18 期间的滑移产生的。我们和其他人最近发现, 5 ‘ poly (a) 领导者赋予 mrna 在感染疫苗病毒 (vacv) 的细胞的翻译优势, 该病毒是天花病毒 19,20的典型成员。
体外转录 rna 基荧光素酶报告法初步开发, 以了解 5 ‘-聚 (a) 引线在猪病毒感染19,21期间 mrna 翻译中的作用。尽管基于粒细胞 dna 的荧光素酶记者检测已被广泛使用, 但有几个缺点会使感染 poxvirus-infected 细胞的结果解释复杂化。首先, 质粒能够在受 vac 感染的22细胞中复制。其次, 神秘转录往往发生在质粒dna 18,23,24。第三, VAC 启动子驱动转录产生聚 (A)-异质长度的引线, 因此在一些实验18中难以控制聚 (A) 引线长度。体外转录 rna 酶报告法规避这些问题, 数据解释很简单。
该方法有四个关键步骤: (1) 聚合酶链反应 (PCR) 生成体外转录的 DNA 模板;(2) 体外转录生成 mRNA;(3) 转染, 将 mRNA 输送到细胞中;(4) 检测荧光素酶活性作为翻译指标 (图 1)。生成的 PCR 放大器包含 5 ‘ 到 3 ‘ 方向中的以下元素: T7-tokor、poly (A) 引线或所需的 5 ‘-utr 序列、萤火虫荧光酶开放阅读框架 (ORF), 然后是聚 (A) 尾。以 PCR 放大器为模板, 利用 T7 聚合酶进行体外转录合成 mRNA。在体外转录过程中,mRNAg 帽或其他盖帽模拟被纳入新合成的 mrna 中。封顶的转录被转移到未经感染或没有感染的 vacv 细胞。细胞裂解液在转染后的所需时间被收集, 以测量表明转染 mRNA 产生蛋白质的荧光素酶活性。本报告法可用于研究 5 ‘-utr、3 ‘-utr 或其他区域 mRNA 中的cis元素的翻译调控。此外, 基于体外转录 rna 的检测方法可用于研究不同的翻译起始机制, 包括与上限相关的起始、与盖帽无关的起始、重新启动和像 RES 这样的内部起始。
Protocol
Representative Results
Discussion
所有四核步骤都是体外转录 rna 基荧光素酶报告法成功的关键。应特别注意引物设计, 特别是 T7 启动子序列。T7 RNA 聚合酶开始转录从在 5 ‘-utr 序列之前添加的 T7 启动子中的下划线第一 g (gGg-5 ‘-utr-aug-)。虽然转录起始位点 (TSS) 从 5 ‘ 端的第一个 G 开始, 但在 T7 启动子区域中, 减少 G 的小于3的数量会减少体外转录的 RNA 产量/输出。在实验中, 我们观察到凝胶纯化 DNA 产物不是最佳的体外转录, 因?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目由国家卫生研究院 (AI128406 www.nih.gov) 资助至 ZY, 部分由约翰逊癌症研究中心 (http://cancer.k-state.edu) 资助, 形式是研究生暑期津贴至 PD。
Materials
| 2X-Q5 Master mix | New England Biolabs | M0492 | High-Fidelity DNA Polymerase used in PCR |
| 3´-O-Me-m7G(5')ppp(5')G RNA Cap Structure Analog | New England Biolabs | S1411L | Anti reverse Cap analog or ARCA |
| Corning 96 Well Half-Area white flat bottom polystyrene microplate | Corning | 3693 | Opaque walled 96 well white plate with solid bottom |
| Dual-Luciferase Reporter Assay System | Promega | E1960 | Dual-Luciferase Assay Kit (DLAK) |
| E.Z.N.A. Cycle Pure Kit | OMEGA BIO-TEK | D6492 | PCR purification kit |
| GloMax Navigator Microplate Luminometer | Promega | GM2010 | Referred as multimode plate reader luminometer |
| HiScribe T7 Quick High Yield RNA synthesis Kit | New England Biolabs | E2050S | In-Vitro transcription kit |
| Lipofectamine 2000 | Thermo Fisher Scientific | 11668019 | Cationic lipid transfection reagent |
| NanoDrop2000 | Thermo Fisher Scientific | ND-2000 | Used to measure DNA and RNA concentration |
| Opti-MEM | Thermo Fisher Scientific | 31985070 | Reduced serum media |
| Purelink RNA Mini Kit | Thermo Fisher Scientific | 12183018A | RNA purification kit |
| Vaccinia Capping System | New England Biolabs | M2080 | Capping system |
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