SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy

  • Nucleic Acids Res. 2019 Apr 8;47(6):e33. doi: 10.1093/nar/gkz049.
Ya Zhao  1  2  3 ,  Yao Zhang  1  4 ,  Yilan Teng  1 ,  Kai Liu  1  5 ,  Yanqing Liu  6 ,  Weihua Li  7 ,  Ligang Wu  1
Affiliations
  • 1. State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, CAS Center for Excellence in Molecular Cell Science, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; University of Chinese Academy of Sciences, Shanghai, China.
  • 2. Institute of Translational Medicine, School of Medicine, Yangzhou University, Yangzhou, China.
  • 3. Jiangsu Key Laboratory of Experimental & Translational Non-coding RNA Research, Yangzhou University, Yangzhou, China.
  • 4. NHC key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), pharmacy school, Fudan University, Shanghai, China.
  • 5. School of Life Sciences, Shanghai University, Shanghai, China.
  • 6. Institute of Integrated Traditional Chinese and Western Medicine, School of Medicine, Yangzhou University, Yangzhou, China.
  • 7. NHC key Laboratory of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Fudan University, Shanghai, China.
PMID: 30715466 DOI: 10.1093/nar/gkz049
Abstract

UV crosslinking and immunoprecipitation (CLIP) coupled with high-throughput Sequencing is the most state-of-the-art technology to characterize protein-RNA interactions, yet only a small portion of RNA-binding proteins (RBPs) have been studied by CLIP due to its complex procedures and high level of false-positive signals. Herein, we report a SpyCLIP method that employs a covalent linkage formed between the RBP-fused SpyTag and SpyCatcher, which can withstand the harshest washing conditions for removing nonspecific interactions. Moreover, SpyCLIP circumvents the radioactive labeling and PAGE-membrane purification steps, and the whole procedure can be performed on beads and is readily amenable to automation. We investigated multiple RBPs by SpyCLIP and generated high-quality RNA binding maps with significantly improved reproductivity and accuracy. Therefore, the small tag size and convenient protocol of SpyCLIP provides a robust method for both routine characterization and high-throughput studies of protein-RNA interactions.

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