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

HIV-1初级转录物反向剪接产生的环状RNA的功能

• 批准号: 10481143
• 负责人: MASSIMO CAPUTI
• 金额: $ 22.88万
• 依托单位: FLORIDA ATLANTIC UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10481143
• 关键词: Adherence; Antisense Oligonucleotides; Autoimmune Diseases; Biogenesis; Biological Assay; CD4 Positive T Lymphocytes; Cardiovascular Diseases; Cell Differentiation process; Cell Line; Cell Proliferation; Cells; Chromatin; Complex; DNA Viruses; Development; Down-Regulation; Drug resistance; Event; Exons; Family; Gene Expression; Generations; Genetic Transcription; Genome; HIV; HIV-1; Half-Life; Herpesviridae; Highly Active Antiretroviral Therapy; Human; Human Herpesvirus 8; Human Papillomavirus; Immune response; Immunologic Deficiency Syndromes; Infection; Interruption; Kaposi Sarcoma; Leukocytes; Life; Link; Malignant Neoplasms; Messenger RNA; MicroRNAs; Modeling; Names; Neoplasm Metastasis; Papillomavirus; Patients; Pattern; Plasmids; Play; Porifera; Protein Isoforms; Proteins; Proviruses; RNA; RNA Binding; RNA Splice Sites; RNA Splicing; RNA Viruses; RNA-Binding Proteins; Reporting; Research; Ribonuclease H; Role; Series; Structure; T-Lymphocyte; Time; Toxic effect; Transcript; Translating; Tumor Suppression; Untranslated RNA; Viral; Viral Cytopathogenic Effect; Viral Genome; Viral reservoir; Virus; Virus Diseases; Virus Latency; Virus Replication; circular RNA; conditioning; cost; design; gain of function; latent infection; loss of function; mRNA Precursor; nervous system disorder; response; transcriptome sequencing

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.

项目摘要 圆形RNA（CIRCRNA）是一个非编码RNA家族，源自非典型剪接 事件，名为Backsplicing，其中RNA转录本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，共价链接到形成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。 Circrnas充当mirnas海绵，因此 抑制其功能，并与RNA结合蛋白（RBP）相互作用，调节其可用性， 本地化和活动。还显示了一些circrnas调节转录，可以是 翻译成短蛋白。在DNA病毒中已经鉴定 疱疹病毒和乳头状膜科家族。这些病毒circrnas已显示出促进细胞 通过充当具有肿瘤抑制功能的miRNA的海绵，增殖和转移。迄今为止， 尚未表征由RNA病毒编码的Circrna。 对复杂HIV-1剪接模式的分析表明，该病毒有可能生成 至少15个不同的circrnas。我们设计了几组发散的PCR引物，以特别放大 预测的HIV CircRNA同工型。设计的底漆被设计为朝相反的方向扩展 来自线性转录本的模板，但延伸到每个模板并放大圆形转录本。我们能够 通过测序15个不同的Circrna同工型，从病毒的后插图产生并确认 前mRNA。鉴于circrnas的半衰期（是线性RNA的4-5倍），这是合理的艾滋病毒 衍生的CIRCRNA通过调节感染细胞来调节病毒复制和感染。在这项研究中，我们 提议定义病毒产生的圆形RNA分子的精确量和组成 在原代T细胞和HIV潜伏期模型中，利用CircRNA同工型特异性QPCR和RNA的组合 测序测定法。我们还将确定HIV-1 ciRCRNA在利用这两者的病毒复制中的作用， 功能分析的增益和丢失。