Footprinting SHAPE-eCLIP Reveals Transcriptome-wide Hydrogen Bonds at RNA-Protein Interfaces

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Understanding RNA-binding protein (RBP) interactions is crucial for gene expression regulation.
• Identifying the precise location and mechanism of RBP binding on RNA is a key challenge.

Purpose of the Study:

• To develop and validate a transcriptome-wide method for mapping RBP footprints on RNA.
• To precisely detect nucleobases involved in protein-RNA hydrogen bonding.

Main Methods:

• Application of selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) on in vivo transcripts.
• Comparison of SHAPE reactivity on native transcripts versus protein-absent transcripts to identify fSHAPE footprints.
• Integration of fSHAPE with enhanced crosslinking and immunoprecipitation (eCLIP) for specific RBP-RNA complex analysis.

Main Results:

• fSHAPE accurately identifies nucleobases that form hydrogen bonds with proteins.
• fSHAPE patterns successfully predict binding sites for known RBPs, including novel loci.
• Combined fSHAPE and eCLIP enable detailed interrogation of specific RNA-protein complexes, like histone stem-loop interactions.

Conclusions:

• fSHAPE provides a powerful tool for transcriptome-wide mapping of RNA-protein interactions.
• This technology enhances the understanding of gene expression regulation by specific RBPs.
• The integration of fSHAPE with other methods expands the study of cellular RNA interactions.