Scholarlink

<p>RNA binding proteins (RBPs) are multi-faceted proteins that interact with transcripts in various RNA driven processes and functions. However, in situ covalent capture techniques for screening authentic RNA substrates of RBPs remain challenging in terms of reproducibility, specificity, and sensitivity. Here, we developed CuCLIP-seq (<Emphasis Type="Underline">Cu</Emphasis>AAC-<Emphasis Type="Underline">C</Emphasis>ross<Emphasis Type="Underline">l</Emphasis>inking and <Emphasis Type="Underline">I</Emphasis>mmuno<Emphasis Type="Underline">p</Emphasis>recipitation <Emphasis Type="Underline">Seq</Emphasis>uencing), an alternative in situ covalent capture sequencing method which utilizes CuAAC reaction to crosslink RBP-RNA between azido groups of RBPs and ethynyl groups of RNA molecules, followed by streptavidin-mediated enrichment of RNA substrates and high-throughput sequencing. We demonstrate the reliability of CuCLIP-seq by identifying substrate RNAs of several RBPs, including PTBP1, ADAR2, SRSF2, HNRNPA1, and PINX1, especially for capturing low-abundance targets. Additionally, this approach is authenticated by specifically resolving the alternative splicing transcripts targeted by PTBP1 and RNA targets by PINX1. Thus, this technique offers a sensitive and specific approach for detecting RBP substrates with high reproducibility, potential scalability, and wide applicability.</p>

错误：搜索内容不能为空，请输入英文关键词
错误：关键词超出字数限制，请精简
高级检索

Novel CuCLIP-seq for in situ covalently captured protein-binding RNAs

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年4月24日

查看全文
  • Xing Wang,
  • Meng-Ke Wang,
  • Bo-Fei Yao,
  • Chun-Chun Gao,
  • Han-Fei Jin,
  • Yu-Sheng Chen,
  • Er-Zhong Wu,
  • Ang Li,
  • Ya-Jing Hao,
  • Yong-Liang Zhao,
  • Yun-Gui Yang,
  • Ying Yang

摘要

RNA binding proteins (RBPs) are multi-faceted proteins that interact with transcripts in various RNA driven processes and functions. However, in situ covalent capture techniques for screening authentic RNA substrates of RBPs remain challenging in terms of reproducibility, specificity, and sensitivity. Here, we developed CuCLIP-seq (CuAAC-Crosslinking and Immunoprecipitation Sequencing), an alternative in situ covalent capture sequencing method which utilizes CuAAC reaction to crosslink RBP-RNA between azido groups of RBPs and ethynyl groups of RNA molecules, followed by streptavidin-mediated enrichment of RNA substrates and high-throughput sequencing. We demonstrate the reliability of CuCLIP-seq by identifying substrate RNAs of several RBPs, including PTBP1, ADAR2, SRSF2, HNRNPA1, and PINX1, especially for capturing low-abundance targets. Additionally, this approach is authenticated by specifically resolving the alternative splicing transcripts targeted by PTBP1 and RNA targets by PINX1. Thus, this technique offers a sensitive and specific approach for detecting RBP substrates with high reproducibility, potential scalability, and wide applicability.