Protocol for capturing the RNA-binding proteome from plants using orthogonal organic phase separation.

Area of Science:

• Molecular Biology
• Proteomics
• Plant Science

Background:

• RNA-binding proteins (RBPs) are crucial regulators of RNA processing, including synthesis, function, transport, and decay.
• Understanding RBP interactions with RNA is vital for deciphering gene regulation.
• Existing methods for isolating RNA-protein complexes can be limiting.

Purpose of the Study:

• To present a novel protocol for the mass isolation of RNA-binding proteins (RBPs) crosslinked to RNA.
• To establish a strategy for subsequent proteomics analysis of these complexes.
• To provide a method applicable to various plant tissues.

Main Methods:

• In vivo UV crosslinking to stabilize RNA-protein interactions.
• Tissue lysis and fractionation to prepare samples.
• Purification of crosslinked RNA-protein adducts using organic solvents.
• Proteomics analysis to identify bound proteins.

Main Results:

• A comprehensive protocol for en masse isolation of crosslinked RNA-protein complexes was successfully developed.
• The protocol enables subsequent proteomics analysis to identify RBPs.
• Demonstrated applicability in Nicotiana benthamiana leaves, with potential for broader plant applications.

Conclusions:

• The developed protocol offers an effective method for isolating RNA-binding proteins (RBPs) crosslinked to RNA.
• This technique facilitates large-scale proteomics studies of RNA-protein interactions in plants.
• The protocol is adaptable for diverse plant species and tissues, advancing RBP research.