Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.
Stem Cell Program, University of California San Diego, La Jolla, CA, USA.
Nat Methods. 2021 May;18(5):507-519. doi: 10.1038/s41592-021-01128-0. Epub 2021 May 7.
RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
RNA 结合蛋白 (RBPs) 是基因表达和 RNA 处理的关键调控因子，对于基因功能至关重要。然而，单个细胞中 RBP 调节的动态性尚不清楚。为了解决这一理解上的空白，我们开发了 STAMP（通过 APOBEC 介导的分析来调查靶标），它可以有效地检测 RBP-RNA 相互作用。STAMP 不依赖于紫外线交联或免疫沉淀，并且与单细胞捕获相结合，可在单个、混合实验中识别多个 RBP 和细胞类型的 RBP 特异性和细胞类型特异性 RNA-蛋白质相互作用。将 STAMP 与长读测序配对，以特定于异构体的方式产生 RBP 靶标位点。最后，Ribo-STAMP 利用小核糖体亚基在单细胞中测量全转录组核糖体关联。STAMP 使我们能够以前所未有的细胞分辨率研究 RBP-RNA 互作组和翻译景观。
