Design of synthetic RNase-resistant RNA structures and their integration in novel riboswitch constructs to regulate mRNA stability

黄病毒是一种单链RNA病毒，其基因组RNA两侧是高度结构化的非翻译区(UTRs)。在3‘-UTR中，进化上保守的RNA元件，称为外切核酸酶抗性RNA(XrRNAs)，能够保护下游区域免受Xrn1和相关的5’-3‘-外切酶的外切核降解，导致稳定的衰退中间产物在感染细胞中积累为长的非编码RNA(LncRNA)物种。这些病毒lncRNAs，也称为亚基因组黄病毒RNAs(SfRNAs)，介导这些病毒的致病作用。我们将利用生物信息学方法更好地了解xrRNAs的2D和3D结构，这将使我们能够设计具有不同能力的合成xrRNA元件，以防止核外降解。设计的元件将在体内和体外通过将它们整合到报告mRNAs的5‘-UTR中来研究它们的外切酶停滞能力，报告mRNAs的开放阅读框架(ORF)是基于内部核糖体进入位点(IRES)翻译的。这种保护的有效性将通过测定信使核糖核酸的半衰期来评估。此外，我们将利用从设计的xrRNA获得的知识来将RNA适配子结构域与兼容的xrRNA结构整合在一起。通过将xrRNA与适体结合，我们将设计出一种核糖开关，可以在存在(或不存在)特定配体的情况下形成保护性结构。这些结构将允许我们在配基络合时稳定(开)或破坏(关开)特定的mRNA。通过使用显示出不同能力的xrRNA元件来拖延外切核酸酶，这将使我们能够修改单个转录本的半衰期。这种对RNA稳定性的诱导调节代表了合成生物学中的一种新方法，它允许对mRNAs和lncRNAs进行细粒度的调节，其半衰期由Xrn1样外切核酸酶决定。

Flaviviruses are single-stranded RNA viruses, whose genomic RNA is flanked by heavily structured untranslated regions (UTRs). Within the 3’-UTR, evolutionarily conserved RNA elements, referred to as exoribonuclease-resistant RNAs (xrRNAs), are capable of protecting downstream regions from exonucleolytic degradation by Xrn1 and related 5’-3’-exonucleases, resulting in the production of stable decay intermediates that accumulate as long non-coding RNA (lncRNA) species in infected cells. These viral lncRNAs, also termed subgenomic flavivirus RNAs (sfRNAs), mediate pathogenicity of these viruses. We will employ bioinformatics methods to better understand the 2D and 3D structure of xrRNAs, which will enable us to design synthetic xrRNA elements with varying capacity to protect against exonucleolytic degradation. Designed elements will be studied in vivo and in vitro for their exonuclease-stalling capacity by integrating them in the 5’-UTR of reporter mRNAs whose open reading frame (ORF) is translated based on an internal ribosome entry site (IRES). The efficiency of this kind of protection will be assessed by determining mRNA half-lives. Moreover, we will employ knowledge acquired with designed xrRNAs to integrate RNA aptamer domains with compatible xrRNA structures. By combining xrRNAs with aptamers we will devise riboswitches that can form a protective structure in response to presence (or absence) of a specific ligand. These constructs will allow us to stabilize (ON switch) or destabilize (OFF switch) a specific mRNA upon ligand complexation. By using xrRNA elements that exhibit different capacities to stall exoribonucleases, this will enable us to modify the half-lives of individual transcripts. This kind of inducible regulation of RNA stability represents a novel approach in synthetic biology that allows for fine-grained modulation of both mRNAs and lncRNAs, whose half-lives are determined by Xrn1-like exoribonucleases.