Department of Molecular Biology and Microbiology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, OH, USA.
Methods Mol Biol. 2024;2807:209-227. doi: 10.1007/978-1-0716-3862-0_15.
The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N-methyladenosine (mA). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map mA at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for mA also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.
HIV RNA 的转录后加工和化学修饰是 HIV 病毒学中研究不足的方面，主要是由于准确绘制和量化 RNA 修饰的能力有限。修饰特异性抗体或修饰敏感内切酶与短读长 RNA 测序技术相结合，可对 HIV RNA 的重要调控修饰（如 N6-甲基腺苷（m6A））进行低分辨率或有限的作图。然而，需要 HIV 转录本上这些位点的高分辨率图谱，才能深入了解其作用机制。最近，新的测序技术使这成为可能。在这里，我们描述了使用 Oxford Nanopore Technologies 测序仪对 HIV 转录本进行直接 RNA 测序，并使用该技术以接近单核苷酸分辨率对 m6A 进行作图。该技术还具有识别长 RNA 读段的剪接变体的能力，因此可以提供区分重叠剪接变体的高分辨率 RNA 修饰图谱。此处概述的 m6A 方案也为研究在纳米孔平台上检测到的任何其他 RNA 修饰提供了强大的范例。
