Department of Chemistry, University of North Carolina, Chapel Hill, NC, USA.
Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA.
Nat Biotechnol. 2021 Mar;39(3):347-356. doi: 10.1038/s41587-020-0709-7. Epub 2020 Oct 19.
RNA-protein interaction networks govern many biological processes but are difficult to examine comprehensively. We devised ribonucleoprotein networks analyzed by mutational profiling (RNP-MaP), a live-cell chemical probing strategy that maps cooperative interactions among multiple proteins bound to single RNA molecules at nucleotide resolution. RNP-MaP uses a hetero-bifunctional crosslinker to freeze interacting proteins in place on RNA and then maps multiple bound proteins on single RNA strands by read-through reverse transcription and DNA sequencing. RNP-MaP revealed that RNase P and RMRP, two sequence-divergent but structurally related non-coding RNAs, share RNP networks and that network hubs define functional sites in these RNAs. RNP-MaP also identified protein interaction networks conserved between mouse and human XIST long non-coding RNAs and defined protein communities whose binding sites colocalize and form networks in functional regions of XIST. RNP-MaP enables discovery and efficient validation of functional protein interaction networks on long RNAs in living cells.
RNA 与蛋白质相互作用网络调控着许多生物过程，但全面研究这些网络具有一定难度。我们设计了一种通过突变分析（RNP-MaP）来解析核糖核蛋白网络的方法，这是一种活细胞化学探测策略，可以在核苷酸分辨率上绘制与单个 RNA 分子结合的多个蛋白质之间的合作相互作用。RNP-MaP 使用一种杂双功能交联剂将相互作用的蛋白质固定在 RNA 上，然后通过通读逆转录和 DNA 测序来绘制单个 RNA 链上的多个结合蛋白。RNP-MaP 揭示了 RNase P 和 RMRP 这两种序列不同但结构相关的非编码 RNA 共享 RNP 网络，而网络枢纽则定义了这些 RNA 中的功能位点。RNP-MaP 还鉴定了小鼠和人类 XIST 长非编码 RNA 之间保守的蛋白质相互作用网络，并定义了结合位点共定位并在 XIST 功能区域形成网络的蛋白质群落。RNP-MaP 能够在活细胞中发现和有效验证长 RNA 上的功能蛋白质相互作用网络。
