Lee Nara
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Methods Mol Biol. 2025;2890:211-224. doi: 10.1007/978-1-0716-4326-6_11.
With the advent of next-generation sequencing, a plethora of techniques have been developed to uncover nucleic acid interactions with unprecedented resolution. For example, UV-crosslinking and immunoprecipitation (CLIP) assays have been coupled to deep-sequencing to identify RNA-protein interactions and precisely map the RNA footprint regions. Here, we describe a CLIP-based technique that allows the genome-wide mapping of influenza nucleoprotein (NP)-binding sites to genomic viral RNA (vRNA). This method has been applied to show that the influenza viral genome is not uniformly coated with nucleoprotein, but instead enriched in certain regions and depleted in others. As subtle changes in genome sequence have been shown to globally alter NP-binding sites, this technique will be useful to deduce how different strains adjust their genome organization and what parameters govern NP-vRNA interactions.
随着下一代测序技术的出现，已经开发出大量技术以空前的分辨率揭示核酸相互作用。例如，紫外线交联免疫沉淀（CLIP）分析已与深度测序相结合，以鉴定RNA-蛋白质相互作用并精确绘制RNA足迹区域。在此，我们描述了一种基于CLIP的技术，该技术可在全基因组范围内将流感核蛋白（NP）结合位点定位到基因组病毒RNA（vRNA）。该方法已被用于表明流感病毒基因组并非均匀地被核蛋白覆盖，而是在某些区域富集而在其他区域耗尽。由于已表明基因组序列的细微变化会全局改变NP结合位点，因此该技术将有助于推断不同毒株如何调整其基因组组织以及哪些参数控制NP-vRNA相互作用。
