Department of Biological Sciences, Auburn University, Auburn, Alabama 36849, USA.
RNA. 2021 Sep;27(9):1102-1125. doi: 10.1261/rna.078777.121. Epub 2021 Jun 29.
Polyadenylated nuclear (PAN) RNA is a long noncoding transcript involved in Kaposi's sarcoma-associated herpesvirus (KSHV) lytic reactivation and regulation of cellular and viral gene expression. We have previously shown that PAN RNA has dynamic secondary structure and protein binding profiles that can be influenced by epitranscriptomic modifications. -methyladenosine (mA) is one of the most abundant chemical signatures found in viral RNA genomes and virus-encoded RNAs. Here, we combined antibody-independent next-generation mapping with direct RNA sequencing to address the epitranscriptomic status of PAN RNA in KSHV infected cells. We showed that PAN mA status is dynamic, reaching the highest number of modifications at the late lytic stages of KSHV infection. Using a newly developed method, termed elenium-modified deoxythymidine triphosphate (SedTTP)-reverse transcription (RT) and igation ssisted CR analysis of mA (SLAP), we gained insight into the fraction of modification at identified sites. By applying comprehensive proteomic approaches, we identified writers and erasers that regulate the mA status of PAN, and readers that can convey PAN mA phenotypic effects. We verified the temporal and spatial subcellular availability of the methylome components for PAN modification by performing confocal microscopy analysis. Additionally, the RNA biochemical probing (SHAPE-MaP) outlined local and global structural alterations invoked by mA in the context of full-length PAN RNA. This work represents the first comprehensive overview of the dynamic interplay that takes place between the cellular epitranscriptomic machinery and a specific viral RNA in the context of KSHV infected cells.
多聚腺苷酸化核 (PAN) RNA 是一种长非编码转录本，参与卡波西肉瘤相关疱疹病毒 (KSHV) 的裂解激活，以及细胞和病毒基因表达的调控。我们之前已经表明，PAN RNA 具有动态的二级结构和蛋白结合谱，这些结构和谱可以受到转录后修饰的影响。N6-甲基腺苷 (m6A) 是在病毒 RNA 基因组和病毒编码的 RNA 中发现的最丰富的化学特征之一。在这里，我们结合了非抗体依赖的下一代图谱技术和直接 RNA 测序，以确定 KSHV 感染细胞中 PAN RNA 的转录后修饰状态。我们表明，PAN m6A 状态是动态的，在 KSHV 感染的晚期裂解阶段达到了最多的修饰。我们使用一种新开发的方法，称为硒代修饰的脱氧胸苷三磷酸 (SedTTP)-逆转录 (RT) 和连接辅助 m6A 分析 (SLAP)，深入了解鉴定位点的修饰分数。通过应用全面的蛋白质组学方法，我们鉴定了调节 PAN m6A 状态的 writers 和 erasers，以及可以传递 PAN m6A 表型效应的 readers。我们通过进行共聚焦显微镜分析，验证了甲基组成分用于 PAN 修饰的时空亚细胞可用性。此外，RNA 生化探测 (SHAPE-MaP) 概述了 m6A 在全长 PAN RNA 背景下引起的局部和全局结构改变。这项工作代表了在 KSHV 感染细胞中，细胞转录后修饰机制与特定病毒 RNA 之间动态相互作用的首次全面概述。
