FLAG Immunoprecipitation-Based Mapping of the In Vivo Assembled Spliceosomal C* Complex

Area of Science:

• Molecular Biology
• Biochemistry
• RNA Biology

Background:

• Pre-mRNA splicing is a crucial process in gene expression, catalyzed by the dynamic spliceosome.
• The spliceosome undergoes significant rearrangements, forming various intermediate complexes (A, B, B^act, C, C*, P).
• Studying these transient spliceosomal intermediates is challenging due to their instability and rapid interconversion.

Purpose of the Study:

• To develop a novel strategy for enriching and studying specific intermediate spliceosomal complexes.
• To investigate the composition of the spliceosomal C* complex using proximity-dependent biotinylation.

Main Methods:

• Utilized a modified MINX splicing substrate with a 3' splice site mutation to arrest spliceosomes at the C* complex stage.
• Combined BioID proximity labeling with MS2 coat protein-mediated RNA tagging and FLAG-MS2 RNP immunoprecipitation.
• Analyzed captured proximal proteins using mass spectrometry to identify spliceosome components.

Main Results:

• Successfully captured and enriched the spliceosomal C* complex using the described strategy.
• Identified proteins proximal to the C* complex, providing insights into its composition.
• Demonstrated the utility of combining BioID with splicing assays for studying spliceosome dynamics.

Conclusions:

• The developed method enables the isolation and characterization of specific spliceosomal intermediates.
• This approach facilitates the study of spliceosome assembly, dynamics, and function.
• Provides a valuable tool for dissecting the molecular mechanisms of pre-mRNA splicing.