Using hiCLIP to identify RNA duplexes that interact with a specific RNA-binding protein

Area of Science:

• Molecular Biology
• Genetics
• Bioinformatics

Background:

• RNA structure is crucial for gene expression regulation, primarily through interactions with RNA-binding proteins (RBPs).
• Identifying RNA-protein interactions and RNA duplexes is essential for understanding gene regulation.
• Existing methods may struggle to identify complex or computationally challenging RNA duplexes.

Purpose of the Study:

• To detail the RNA hybrid and individual-nucleotide resolution UV cross-linking and immunoprecipitation (hiCLIP) protocol.
• To provide a streamlined data analysis pipeline using the 'hiclipr' R package.
• To enable accurate identification of RNA duplexes bound by specific RBPs.

Main Methods:

• hiCLIP involves in vivo cross-linking, RNA digestion, immunoprecipitation, linker ligation, reverse transcription, and high-throughput sequencing.
• The method specifically identifies RNA duplexes bound by a target RNA-binding protein.
• Sequenced RNA arms are mapped to a reference transcriptome to pinpoint duplex locations.

Main Results:

• hiCLIP can identify all types of RNA duplexes, including intermolecular and long-range intramolecular structures.
• The use of a linker adaptor ensures unambiguous identification of RNA duplexes.
• The hiCLIP protocol and 'hiclipr' R package offer a streamlined approach for analyzing RNA-protein interactions.

Conclusions:

• hiCLIP is a powerful transcriptome-wide method for mapping RNA duplexes bound by RBPs.
• The protocol facilitates the discovery of complex RNA structures influencing gene expression.
• The described procedure and analysis tools simplify the study of RNA-protein interactions.