Protocol to isolate RBP-mRNA complexes using RNA-CLIP and examine target mRNAs.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• RNA-binding proteins (RBPs) are crucial regulators of gene expression, influencing cellular functions through interactions with target messenger RNA (mRNA) molecules.
• Understanding RBP-mRNA interactions is vital for deciphering complex biological processes, including development, physiology, and disease pathogenesis.

Purpose of the Study:

• To present a detailed protocol for isolating RNA-binding protein-mRNA complexes using RNA-induced crosslinking followed by immunoprecipitation (RNA-CLIP).
• To enable the examination of target mRNAs associated with ribosomal populations, providing insights into translational regulation.
• To facilitate the identification of specific RBPs and their cognate RNA targets across diverse physiological and pathological conditions.

Main Methods:

• The protocol involves RNA-induced crosslinking to stabilize RBP-mRNA interactions.
• Immunoprecipitation is employed to isolate specific RBP-mRNA complexes.
• RNA sequencing is used to identify and quantify target mRNAs associated with the captured RBPs.

Main Results:

• The described RNA-CLIP protocol allows for the isolation of RBP-mRNA complexes from various tissue sources, including liver and small intestine.
• The method is also applicable to populations of primary cells, such as hepatocytes.
• The protocol successfully identifies specific RBPs and their associated RNA targets relevant to different biological states.

Conclusions:

• This protocol provides a robust method for studying RBP-mRNA interactions in multicellular tissues and primary cell populations.
• It offers a valuable tool for investigating the roles of RBPs in regulating gene expression during development, normal physiology, and disease.
• The technique aids in understanding the intricate network of RNA-protein interactions governing cellular function.