Work

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

Year: 2025

Type: article

Abstract: Pre-mRNA splicing is catalyzed by the ribonucleoprotein (RNP) complex known as the spliceosome. The spliceosomes are dynamic and undergo constant rearrangement, leading to the formation of the different spliceosomal complexes A, B, Bact, C, C*, and P. Isolation of the spliceosomal complex at a specific intermediate stage requires a means to enrich it. This study describes a strategy for studying intermediate spliceosomal complexes by combining BioID with splicing assays. The MINX splicing substrate with a mutation at the 3′ splice site was utilized to arrest and capture the spliceosomal C* complex before the second catalytic step of splicing. The splicing substrate also contains binding sites for the MS2 coat protein, which facilitates the pull-down of assembled complex by FLAG-MS2-tagged RNP immunoprecipitation and determines the captured proximal proteins by mass spectrometry.

Source: International Journal of Molecular Sciences

DOI: 10.3390/ijms26209914

Authors: Sweta Kumari, Kusum K. Singh

Institution: Indian Institute of Technology Guwahati

Language: English

Topic: RNA Research and Splicing

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,499

Funder: Science and Engineering Research Board