State Key Laboratory of Proteomics, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing Proteome Research Center, Beijing 102206, China.
Analyst. 2021 Feb 22;146(4):1188-1197. doi: 10.1039/d0an02305a.
RNA-protein interactions play important roles in almost every step of the lifetime of RNAs, such as RNA splicing, transporting, localization, translation and degradation. Post-translational modifications, such as O-GlcNAcylation and phosphorylation, and their "cross-talk" (OPCT) are essential to the activity and function regulation of RNA-binding proteins (RBPs). However, due to the extremely low abundance of O-GlcNAcylation and the lack of RBP-targeted enrichment strategies, large-scale simultaneous profiling of O-GlcNAcylation and phosphorylation on RBPs is still a challenging task. In the present study, we developed a tandem enrichment strategy combining metabolic labeling-based RNA tagging for selective purification of RBPs and HILIC-based enrichment for simultaneous O-GlcNAcylation and phosphorylation profiling. Benefiting from the sequence-independent RNA tagging by ethynyluridine (EU) labeling, 1115 RBPs binding to different types of RNAs were successfully enriched and identified by quantitative mass spectrometry (MS) analysis. Further HILIC enrichment on the tryptic-digested RBPs and MS analysis led to the first large-scale identification of O-GlcNAcylation and phosphorylation in the RNA-binding proteome, with 461 O-GlcNAc peptides corresponding to 300 RBPs and 671 phosphopeptides corresponding to 389 RBPs. Interestingly, ∼25% RBPs modified by two PTMs were found to be related to multiple metabolism pathways. This strategy has the advantage of high compatibility with MS and provides peptide-level evidence for the identification of O-GlcNAcylated RBPs. We expect it will support simultaneous mapping of O-GlcNAcylation and phosphorylation on RBPs and facilitate further elucidation of the crucial roles of OPCT in the function regulation of RBPs.
RNA 与蛋白质的相互作用在 RNA 的几乎所有生命周期步骤中都发挥着重要作用，例如 RNA 的剪接、运输、定位、翻译和降解。翻译后修饰（如 O-GlcNAc 化和磷酸化）及其“串扰”（OPCT）对于 RNA 结合蛋白（RBPs）的活性和功能调节至关重要。然而，由于 O-GlcNAc 化的丰度极低，并且缺乏针对 RBP 的靶向富集策略，因此大规模同时对 RBPs 上的 O-GlcNAc 化和磷酸化进行分析仍然是一项具有挑战性的任务。在本研究中，我们开发了一种串联富集策略，将基于代谢标记的 RNA 标记用于选择性纯化 RBPs 与亲水相互作用色谱（HILIC）富集相结合，以同时对 O-GlcNAc 化和磷酸化进行分析。得益于通过炔基尿嘧啶（EU）标记实现的序列无关的 RNA 标记，成功地通过定量质谱（MS）分析富集和鉴定了 1115 个与不同类型 RNA 结合的 RBPs。进一步对酶切的 RBPs 进行 HILIC 富集和 MS 分析，首次大规模鉴定了 RNA 结合蛋白组中的 O-GlcNAc 化和磷酸化，鉴定到 461 个 O-GlcNAc 肽对应 300 个 RBPs 和 671 个磷酸肽对应 389 个 RBPs。有趣的是，发现约 25%的同时被两种 PTM 修饰的 RBPs 与多种代谢途径有关。该策略与 MS 具有高度兼容性，并为鉴定 O-GlcNAc 化的 RBPs 提供了肽水平的证据。我们期望它将支持同时对 RBPs 上的 O-GlcNAc 化和磷酸化进行作图，并有助于进一步阐明 OPCT 在 RBPs 功能调节中的关键作用。
