Functions of circular RNAs generated from backsplicing of the HIV-1 primary transcript

项目总结 环状RNA(CircRNAs)是一类起源于非规范剪接的非编码RNA 事件，称为反向剪接，在该事件中，RNA转录本的上游和下游剪接位点变成 共价连接形成RNA的闭合环，而不是典型的mRNA转录本的线性结构。 细胞CircRNA在发育、细胞分化、增殖中发挥作用，并与癌症有关， 神经疾病、自身免疫和心血管疾病。CircRNA充当miRNAs海绵，因此 抑制它们的功能，并通过与RNA结合蛋白(RBPs)相互作用，调节它们的可用性， 本地化和活跃性。一些CircRNA也被证明可以调节转录，并可以 转化成短蛋白质。已在来自中国的DNA病毒中发现了源于病毒的CircRNA 疱疹病毒科和乳头病毒科。这些病毒环状RNA已被证明能促进细胞 增殖和转移，作为具有肿瘤抑制功能的miRNAs的海绵。到目前为止， 目前还没有鉴定出由RNA病毒编码的CircRNA。 对复杂的HIV-1剪接模式的分析表明，该病毒有可能在 至少15个不同的CircRNA。我们设计了几组不同的聚合酶链式反应引物来特异性地扩增 预测的HIV CircRNA亚型。发散的引物被设计成向相反的方向延伸 模板来自线性转录本，但向每个模板延伸并放大循环转录本。我们能够 通过测序扩增和确认病毒反向剪接产生的15种不同的CircRNA异构体 前信使核糖核酸。鉴于CircRNA的半衰期很长(是线性RNA的4-5倍)，有可能HIV 衍生的CircRNA通过调节受感染的细胞来调节病毒复制和感染。在这项研究中，我们 建议确定病毒产生的环状RNA分子的确切数量和组成 在原代T细胞和利用CircRNA异构体特异性qPCR和RNA组合的HIV潜伏期模型中 测序分析。我们还将利用两者来确定HIV-1 CircRNA在病毒复制中的作用， 功能得失分析。

PROJECT SUMMARY Circular RNAs (circRNAs) are a family of non-coding RNAs that originate from a non-canonical splicing event, named backsplicing, in which the upstream and downstream splicing sites of an RNA transcript become covalently linked to form a closed loop of RNA as opposed to the typical linear structure of mRNA transcripts. Cellular circRNAs play a role in development, cell differentiation, proliferation and are associated with cancer, neurological disorders, autoimmune and cardiovascular disease. circRNAs act as miRNAs sponges, thus inhibiting their function, and by interacting with RNA binding proteins (RBPs), modulating their availability, localization and activity. A few circRNAs have been also shown to modulate transcription and can be translated into short proteins. CircRNAs of viral origin have been identified in DNA viruses from the Herpesviridae and Papillomaviridae families. These viral circRNAs have been shown to promote cell proliferation and metastasis by acting as sponges for miRNAs with functions in tumor suppression. To date, no circRNA encoded by an RNA virus has been characterized. Analysis of the complex HIV-1 splicing pattern revealed that this virus has the potential to generate at least 15 distinct circRNAs. We designed several sets of divergent PCR primers to specifically amplify the predicted HIV circRNA isoforms. Divergent primers were designed to extend in opposite directions on templates from linear transcripts but extend towards each and amplify circularized transcripts. We were able to amplify and confirm by sequencing 15 distinct circRNA isoforms generated from backsplicing of the viral pre-mRNA. Given the long half-life of circRNAs (4-5 times the one of linear RNAs) it is plausible that HIV derived circRNAs modulate viral replication and infection by conditioning the infected cells. In this study we propose to define the precise amount and composition of the circular RNA molecules produced by the virus in primary T cells and a HIV latency model utilizing a combination of circRNA isoform specific qPCR and RNA Sequencing assays. We will also determine the role of the HIV-1 circRNAs in viral replication utilizing both, gain and loss of functions assays.