Center for RNA Research, Institute for Basic Science, Seoul 08826, Korea.
School of Biological Sciences, Seoul National University, Seoul 08826, Korea.
Nucleic Acids Res. 2021 Mar 18;49(5):e28. doi: 10.1093/nar/gkaa1194.
RNA-protein interaction is central to post-transcriptional gene regulation. Identification of RNA-binding proteins relies mainly on UV-induced crosslinking (UVX) followed by the enrichment of RNA-protein conjugates and LC-MS/MS analysis. However, UVX has limited applicability in tissues of multicellular organisms due to its low penetration depth. Here, we introduce formaldehyde crosslinking (FAX) as an alternative chemical crosslinking for RNA interactome capture (RIC). Mild FAX captures RNA-protein interaction with high specificity and efficiency in cell culture. Unlike UVX-RIC, FAX-RIC robustly detects proteins that bind to structured RNAs or uracil-poor RNAs (e.g. AGO1, STAU1, UPF1, NCBP2, EIF4E, YTHDF proteins and PABP), broadening the coverage. Applied to Xenopus laevis oocytes and embryos, FAX-RIC provided comprehensive and unbiased RNA interactome, revealing dynamic remodeling of RNA-protein complexes. Notably, translation machinery changes during oocyte-to-embryo transition, for instance, from canonical eIF4E to noncanonical eIF4E3. Furthermore, using Mus musculus liver, we demonstrate that FAX-RIC is applicable to mammalian tissue samples. Taken together, we report that FAX can extend the RNA interactome profiling into multicellular organisms.
RNA-蛋白质相互作用是转录后基因调控的核心。RNA 结合蛋白的鉴定主要依赖于紫外线诱导的交联 (UVX)，随后进行 RNA-蛋白质缀合物的富集和 LC-MS/MS 分析。然而，由于其穿透深度有限，UVX 在多细胞生物的组织中应用有限。在这里，我们引入甲醛交联 (FAX) 作为 RNA 互作组捕获 (RIC) 的替代化学交联方法。在细胞培养中，温和的 FAX 以高特异性和高效率捕获 RNA-蛋白质相互作用。与 UVX-RIC 不同，FAX-RIC 可以稳健地检测到与结构 RNA 或尿嘧啶贫乏 RNA（例如 AGO1、STAU1、UPF1、NCBP2、EIF4E、YTHDF 蛋白和 PABP）结合的蛋白质，从而扩大了覆盖范围。在非洲爪蟾卵母细胞和胚胎中的应用提供了全面而无偏倚的 RNA 互作组，揭示了 RNA-蛋白质复合物的动态重塑。值得注意的是，在卵母细胞到胚胎的转变过程中，翻译机制发生了变化，例如从典型的 eIF4E 到非典型的 eIF4E3。此外，我们使用 Mus musculus 肝脏证明了 FAX-RIC 适用于哺乳动物组织样本。总之，我们报告说 FAX 可以将 RNA 互作组分析扩展到多细胞生物中。
