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. N⁶-methyladenosine (m⁶A) 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 m⁶A status is dynamic, reaching the highest number of modifications at the late lytic stages of KSHV infection. Using a newly developed method, termed selenium-modified deoxythymidine triphosphate (SedTTP)-reverse transcription (RT) and ligation assisted PCR analysis of m⁶A (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 m⁶A status of PAN, and readers that can convey PAN m⁶A 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 m⁶A 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.

中文翻译：

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KSHV 复制背景下多聚腺苷酸化核 (PAN) RNA 及其相关甲基化组的 m6A 景观


多聚腺苷酸核 (PAN) RNA 是一种长非编码转录物，参与卡波西肉瘤相关疱疹病毒 (KSHV) 裂解性再激活以及细胞和病毒基因表达的调节。我们之前已经证明，PAN RNA 具有动态二级结构和蛋白质结合特征，可能受到表观转录组修饰的影响。 N ⁶ -甲基腺苷 (m ⁶ A) 是病毒 RNA 基因组和病毒编码 RNA 中发现的最丰富的化学特征之一。在这里，我们将不依赖于抗体的下一代作图与直接 RNA 测序相结合，以解决 KSHV 感染细胞中 PAN RNA 的表观转录组状态。我们发现 PAN m ⁶ A 状态是动态的，在 KSHV 感染的裂解后期达到最高数量的修饰。使用一种新开发的方法，称为s硒修饰脱氧胸苷三磷酸 (SedTTP) - 逆转录 (RT) 和连接m ⁶ A 的辅助PCR分析 (SLAP)，我们深入了解了已识别位点的修饰比例。通过应用综合蛋白质组学方法，我们确定了调节 PAN m ⁶ A 状态的写入器和擦除器，以及可以表达 PAN m ⁶ A 表型效应的读取器。我们通过共聚焦显微镜分析验证了用于 PAN 修饰的甲基化组成分的时间和空间亚细胞可用性。此外，RNA 生化探测 (SHAPE-MaP) 概述了 m ⁶ A 在全长 PAN RNA 背景下引起的局部和整体结构变化。 这项工作首次全面概述了 KSHV 感染细胞中细胞表观转录组机制与特定病毒 RNA 之间发生的动态相互作用。