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Related Experiment Video

Updated: Oct 14, 2025

Monitoring Protein-RNA Interaction Dynamics In Vivo at High Temporal Resolution Using &#967;CRAC
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CRISPR-based RNA-binding protein mapping in live cells.

Baiwen Chen1, Haiyan Shi2, Jia Zhang1

  • 1ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital,Fudan University, Shanghai, 200031, China.

Biochemical and Biophysical Research Communications
|November 4, 2021
PubMed
Summary

Researchers developed a new CRISPR-based method to identify proteins interacting with specific RNAs in live cells. This technique successfully identified 226 proteins bound to U1 small nuclear RNA, advancing RNA-RBP interaction studies.

Keywords:
RNA-Binding proteinU1 snRNAdCas13a

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Area of Science:

  • Molecular Biology
  • Proteomics
  • Genomics

Background:

  • RNA-binding proteins (RBPs) regulate crucial cellular processes, including RNA metabolism, homeostasis, and viral replication.
  • Understanding specific RNA-RBP interactions is vital, yet current methods have limitations in identifying proteins associated with individual RNAs.
  • The global RNA-binding proteome has been characterized, but detailed analysis of proteins binding to specific RNA molecules remains challenging.

Purpose of the Study:

  • To develop and optimize a novel CRISPR-based RNA interaction proteomics method for live-cell applications.
  • To identify proteins that specifically bind to U1 small nuclear RNA using the developed method.

Main Methods:

  • A CRISPR-based system utilizing dCas13a fused to an HA tag was employed to target specific RNAs guided by gRNA.
  • RNA-protein complexes were crosslinked via UV irradiation after dCas13a binding.
  • Proteins were captured using anti-HA beads, purified, and identified through mass spectrometry.

Main Results:

  • The novel CRISPR-based method was successfully optimized for live-cell RNA interactome capture.
  • Application of the method to U1 small nuclear RNA identified 226 associated proteins.
  • This provides a significant expansion of known proteins interacting with U1 small nuclear RNA.

Conclusions:

  • The developed CRISPR-based RNA interaction proteomics method is effective for identifying specific RNA-binding proteins in live cells.
  • This technique offers a powerful tool for dissecting RNA-RBP interactions, crucial for understanding cellular functions and disease mechanisms.
  • The identification of 226 proteins interacting with U1 small nuclear RNA opens new avenues for research into its regulatory roles.