Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.
Centre for Palaeogenetics, Stockholm, Sweden.
Nat Biotechnol. 2024 Aug;42(8):1296-1302. doi: 10.1038/s41587-023-01951-0. Epub 2023 Sep 21.
MicroRNAs (miRNAs) exert their gene regulatory effects on numerous biological processes based on their selection of target transcripts. Current experimental methods available to identify miRNA targets are laborious and require millions of cells. Here we have overcome these limitations by fusing the miRNA effector protein Argonaute2 to the RNA editing domain of ADAR2, allowing the detection of miRNA targets transcriptome-wide in single cells. miRNAs guide the fusion protein to their natural target transcripts, causing them to undergo A>I editing, which can be detected by sensitive single-cell RNA sequencing. We show that agoTRIBE identifies functional miRNA targets, which are supported by evolutionary sequence conservation. In one application of the method we study microRNA interactions in single cells and identify substantial differential targeting across the cell cycle. AgoTRIBE also provides transcriptome-wide measurements of RNA abundance and allows the deconvolution of miRNA targeting in complex tissues at the single-cell level.
微小 RNA（miRNAs）通过选择靶转录本，对众多生物过程发挥基因调控作用。目前可用的鉴定 miRNA 靶标的实验方法既繁琐又需要数百万个细胞。在这里，我们通过将 miRNA 效应蛋白 Argonaute2 融合到 ADAR2 的 RNA 编辑结构域，克服了这些限制，从而可以在单细胞中对 miRNA 靶标进行全转录组检测。miRNAs 引导融合蛋白与天然靶转录本结合，导致它们发生 A>I 编辑，这可以通过灵敏的单细胞 RNA 测序检测到。我们表明 agoTRIBE 可以识别功能性 miRNA 靶标，这些靶标得到了进化序列保守性的支持。在该方法的一个应用中，我们在单细胞中研究了 microRNA 相互作用，并鉴定出细胞周期中存在大量的靶向差异。agoTRIBE 还提供了全转录组的 RNA 丰度测量，并允许在单细胞水平上对复杂组织中的 miRNA 靶向进行分解。
