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

项目摘要 环状RNA（CircRNA）是一类非编码RNA家族，起源于非典型剪接 事件，称为反向剪接，其中RNA转录物的上游和下游剪接位点成为 与mRNA转录物的典型线性结构相反，RNA的环状结构共价连接以形成RNA的闭环。 细胞circRNA在发育、细胞分化、增殖中起作用，并与癌症相关， 神经系统疾病、自身免疫和心血管疾病。circRNA充当miRNAs海绵，因此 抑制其功能，并通过与RNA结合蛋白（RBP）相互作用，调节其可用性， 定位和活动。一些circRNA也已被证明可以调节转录，并且可以被 翻译成短蛋白质。病毒来源的CircRNA已经在来自以下国家的DNA病毒中被鉴定： 疱疹病毒科和乳头瘤病毒科。这些病毒circRNA已被证明可以促进细胞 通过充当具有肿瘤抑制功能的miRNAs的海绵来抑制增殖和转移。到目前为止， 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，这种病毒有可能在 至少15种不同的circRNA。我们设计了几套不同的PCR引物， 预测HIV circRNA亚型。将发散引物设计成在引物上以相反方向延伸。 模板从线性转录物，但延伸到每个和扩增环化的转录物。我们能够 扩增并通过对病毒反向剪接产生的15种不同的circRNA同种型进行测序进行确认， 前mRNA。考虑到circRNA的半衰期长（线性RNA的4-5倍）， 衍生的circRNA通过调节感染的细胞来调节病毒复制和感染。本研究 我建议确定病毒产生的环状RNA分子的精确数量和组成， 在原代T细胞和HIV潜伏期模型中使用circRNA同种型特异性qPCR和RNA 测序测定。我们还将确定HIV-1 circRNA在病毒复制中的作用， 功能测定的获得和丧失。

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.