N6-methyladenosine and the biology of HIV

 描述(申请人提供)：最近的技术进步表明，m6A甲基化在人类信使核糖核酸和长的非编码RNA中大量存在，并可以指导广泛的细胞过程。HIV基因组包含许多假定的m6A修饰位点，我们的初步实验数据表明，HIV基因组中存在m6A修饰。这项拟议研究的总体目标是确定HIV基因组RNA中m6A修饰的准确位置，并确定这些修饰对HIV生物学的功能影响。在目标1中，我们将应用位点特异性切割和放射性标记，然后连接辅助提取和薄层层析(猩红)，以单核苷酸分辨率定量和精确定位m6A修饰。在目标2中，我们将研究m6A RNA修饰对HIV感染性和病毒产生的影响。此外，我们将通过感染单核细胞来源的树突状细胞来测试HIV中不同的m6A对HIV免疫原性的影响。拟议的实验将表征一种新的HIV RNA的RNA修饰模式。对艾滋病毒m6A修饰的表征及其在艾滋病毒生命周期中的作用的阐明不仅将扩大我们对艾滋病毒的了解，而且还将扩大对其他m6A修饰病毒的了解。

 DESCRIPTION (provided by applicant): Recent technological advances showed that m6A methylation is abundant in human mRNA and long non- coding RNA and can guide a wide range of cellular processes. The HIV genome comprises numerous putative m6A modification sites and our preliminary experimental data show that m6A modifications are present in the HIV genome. The overall goal of the proposed research is to pinpoint the precise sites of m6Amodifications within the HIV genomic RNA and to determine the functional impact of these modifications on HIV biology. In Aim 1 we will apply Site-specific Cleavage And Radioactive-labeling followed by Ligation-assisted Extraction and Thin-layer chromatography (SCARLET), to quantify and precisely locate m6A modifications at a single nucleotide resolution. In Aim 2 we will study the effects of m6A RNA modifications on HIV infectivity and viral production. In addition, we will test the effect of varying m6A in HIV on the immunogenicity of HIV by infecting monocyte derived dendritic cells. The proposed experiments will characterize a novel mode of RNA modification of HIV RNA. The characterization of HIV m6A modifications and its elucidation of its role in the HIV life cycle would not only expand our knowledge of HIV, but also for other viruses that are m6A modified.