Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

作者： Rongbing Huang*1,2, Mengting Han*1,2, Liying Meng1,3, Xing Chen1,2,3,4,5 1College of Chemistry and Molecular Engineering,Peking University, 2Beijing National Laboratory for Molecular Sciences,Peking University, 3Peking-Tsinghua Center for Life Sciences,Peking University, 4Synthetic and Functional Biomolecules Center,Peking University, 5Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education,Peking University

简介：

Overview

This article presents a detailed protocol for the click chemistry-assisted RNA-interactome capture (CARIC) strategy, which identifies proteins binding to both coding and noncoding RNAs. CARIC provides a comprehensive census of RNA-binding proteins, aiding in the understanding of post-transcriptional gene regulation.

Key Study Components

Area of Science

• RNA biology
• Protein-RNA interactions
• Post-transcriptional regulation

Background

• Understanding RNA-binding proteins is crucial for gene regulation.
• CARIC captures proteins bound to various RNA types, including mRNAs and non-coding RNAs.
• This method can be applied across different organisms.
• HeLa cells are commonly used for such studies.

Purpose of Study

• To provide a protocol for identifying RNA-binding proteins.
• To enhance the understanding of RNA-protein interactions.
• To facilitate research in post-transcriptional gene regulation.

Methods Used

• Culturing HeLa cells in supplemented DMEM.
• Using 100 millimolar EU to label RNA.
• Performing click chemistry for RNA-interactome capture.
• Analyzing protein-RNA interactions.

Main Results

• Successful identification of RNA-binding proteins.
• Demonstrated the versatility of CARIC across different RNA types.
• Provided insights into the dynamics of RNA-protein interactions.
• Established a reliable method for future research in RNA biology.

Conclusions

• CARIC is a powerful tool for studying RNA-binding proteins.
• This method can advance the understanding of gene regulation mechanisms.
• Future applications may include various biological contexts and organisms.

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