Department of Biochemistry and Molecular Genetics, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
RNA Bioscience Initiative, University of Colorado Anschutz Medical Campus, Aurora, Colorado.
Curr Protoc. 2022 May;2(5):e424. doi: 10.1002/cpz1.424.
The subcellular localization of specific RNA molecules promotes localized cellular activity across a variety of species and cell types. The misregulation of this RNA targeting can result in developmental defects, and mutations in proteins that regulate this process are associated with multiple diseases. For the vast majority of localized RNAs, however, the mechanisms that underlie their subcellular targeting are unknown, partly due to the difficulty associated with profiling and quantifying subcellular RNA populations. To address this challenge, we developed Halo-seq, a proximity labeling technique that can label and profile local RNA content at virtually any subcellular location. Halo-seq relies on a HaloTag fusion protein localized to a subcellular space of interest. Through the use of a radical-producing Halo ligand, RNAs that are near the HaloTag fusion are specifically labeled with spatial and temporal control. Labeled RNA is then specifically biotinylated in vitro via a click reaction, facilitating its purification from a bulk RNA sample using streptavidin beads. The content of the biotinylated RNA is then profiled using high-throughput sequencing. In this article, we describe the experimental and computational procedures for Halo-seq, including important benchmark and quality control steps. By allowing the flexible profiling of a variety of subcellular RNA populations, we envision Halo-seq facilitating the discovery and further study of RNA localization regulatory mechanisms. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Visualization of HaloTag fusion protein localization Basic Protocol 2: In situ copper-catalyzed cycloaddition of fluorophore via click reaction Basic Protocol 3: In vivo RNA alkynylation and extraction of total RNA Basic Protocol 4: In vitro copper-catalyzed cycloaddition of biotin via click reaction Basic Protocol 5: Assessment of RNA biotinylation by RNA dot blot Basic Protocol 6: Enrichment of biotinylated RNA using streptavidin beads and preparation of RNA-seq library Basic Protocol 7: Computational analysis of Halo-seq data.
特定 RNA 分子的亚细胞定位促进了各种物种和细胞类型的局部细胞活动。这种 RNA 靶向的失调可能导致发育缺陷，并且调节此过程的蛋白质突变与多种疾病有关。然而，对于绝大多数局部化 RNA 来说，其亚细胞靶向的机制尚不清楚，部分原因是难以对亚细胞 RNA 群体进行分析和定量。为了解决这一挑战，我们开发了 Halo-seq，这是一种接近标记技术，可以在几乎任何亚细胞位置标记和分析局部 RNA 含量。Halo-seq 依赖于定位在感兴趣的亚细胞空间的 HaloTag 融合蛋白。通过使用产生自由基的 Halo 配体，靠近 HaloTag 融合蛋白的 RNA 可以被特异性标记，具有空间和时间的控制。然后，通过点击反应在体外特异性地将标记的 RNA 生物素化，便于使用链霉亲和素珠从大量 RNA 样品中纯化。然后使用高通量测序对生物素化 RNA 的含量进行分析。在本文中，我们描述了 Halo-seq 的实验和计算程序，包括重要的基准和质量控制步骤。通过允许对各种亚细胞 RNA 群体进行灵活的分析，我们设想 Halo-seq 将有助于发现和进一步研究 RNA 定位调节机制。© 2022 威立公司。基本方案 1：可视化 HaloTag 融合蛋白定位基本方案 2：通过点击反应在原位铜催化荧光团的环加成基本方案 3：体内 RNA 炔基化和总 RNA 的提取基本方案 4：体外铜催化生物素的点击反应基本方案 5：通过 RNA 点印迹评估 RNA 生物素化基本方案 6：使用链霉亲和素珠从总 RNA 中富集生物素化 RNA 并制备 RNA-seq 文库基本方案 7：Halo-seq 数据的计算分析。
