Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks

•Described is a method to reversibly constrain a nucleic acid-protein complex.•A site-specific N-thioalkyl in the backbone of a nucleic acid is engineered.•A disulfide cross-link is formed between a cysteine sidechain and an N-thioalkyl.•A pre-mRNA and U1 snRNP, an 11-subunit RNA-protein complex, are reversibly trapped.

The spliceosome is a highly dynamic mega-Dalton enzyme, formed in part by assembly of U snRNPs onto its pre-mRNA substrate transcripts. Early steps in spliceosome assembly are challenging to study biochemically and structurally due to compositional and conformational dynamics. We detail an approach to covalently and reversibly constrain or trap non-covalent pre-mRNA/protein spliceosome complexes. This approach involves engineering a single disulfide bond between a thiol-bearing cysteine sidechain and a proximal backbone phosphate of the pre-mRNA, site-specifically modified with an N-thioalkyl moiety. When distance and angle between reactants is optimal, the sidechain will react with the single N-thioalkyl to form a crosslink upon oxidation. We provide protocols detailing how this has been applied successfully to trap an 11-subunit RNA-protein assembly, the human U1 snRNP, in complex with a pre-mRNA.