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Functional analyses of mammalian virus 5’UTR-derived, small RNAs that regulate virus translation
Methods ( IF 4.8 ) Pub Date : 2020-11-01 , DOI: 10.1016/j.ymeth.2020.02.008
Mei-Ling Li 1 , Gary Brewer 1
Affiliation  

Enterovirus A71 (EV-A711) RNA contains an internal ribosomal entry site (IRES) to direct cap-independent translation. IRES-dependent translation requires the host's translation initiation factors and IRES-associated trans-acting factors (ITAFs). We previously showed that hnRNP A1, the mRNA stability factor HuR, and the RISC subunit Argonaute 2 (Ago2) are ITAFs that associate with stem loop II (SL-II) of the IRES and promote IRES-dependent translation. By contrast, the mRNA decay factor AUF1 is a negative-acting ITAF that also binds SL-II. Moreover, the small RNA-processing enzyme Dicer produces at least four virus-derived, small RNAs (vsRNAs 1-4) from the EV-A71 5'UTR in infected cells. One of these, vsRNA1, derived from SL-II, inhibits IRES activity via an unknown mechanism. In vitro RNA-binding assays revealed that vsRNA1 can alter association of Ago2, HuR, and AUF1 with SL-II. This presents a possible mechanism by which vsRNA1 could control association of ITAFs with the IRES and modulate viral translation. Here, we describe methods for functional analyses of vsRNA1-mediated regulation of IRES activity. These methods should be applicable to other virus-derived, small RNAs as well.

中文翻译:

哺乳动物病毒 5'UTR 衍生的调节病毒翻译的小 RNA 的功能分析

肠道病毒 A71 (EV-A711) RNA 包含一个内部核糖体进入位点 (IRES),可指导独立于帽的翻译。IRES 依赖性翻译需要宿主的翻译起始因子和 IRES 相关的反式作用因子 (ITAF)。我们之前表明 hnRNP A1、mRNA 稳定因子 HuR 和 RISC 亚基 Argonaute 2 (Ago2) 是与 IRES 的茎环 II (SL-II) 相关联并促进 IRES 依赖性翻译的 ITAF。相比之下,mRNA 衰减因子 AUF1 是一种负作用的 ITAF,它也与 SL-II 结合。此外,小 RNA 加工酶 Dicer 从受感染细胞中的 EV-A71 5'UTR 产生至少四种病毒衍生的小 RNA(vsRNAs 1-4)。其中之一,来自 SL-II 的 vsRNA1,通过未知机制抑制 IRES 活性。体外 RNA 结合分析表明 vsRNA1 可以改变 Ago2、HuR 和 AUF1 与 SL-II 的关联。这提出了一种可能的机制,通过该机制,vsRNA1 可以控制 ITAF 与 IRES 的关联并调节病毒翻译。在这里,我们描述了对 vsRNA1 介导的 IRES 活性调节的功能分析方法。这些方法也应适用于其他病毒衍生的小 RNA。
更新日期:2020-11-01
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