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Updated: Feb 27, 2026

Combining Non-reducing SDS-PAGE Analysis and Chemical Crosslinking to Detect Multimeric Complexes Stabilized by Disulfide Linkages in Mammalian Cells in Culture
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Reversibly constraining spliceosome-substrate complexes by engineering disulfide crosslinks.

Patrick McCarthy1, Erin Garside2, Yonatan Meschede-Krasa1

  • 1Department of Biochemistry, Brandeis University, Waltham, MA, USA.

Methods (San Diego, Calif.)
|June 27, 2017
PubMed
Summary
This summary is machine-generated.

Researchers developed a new method to trap spliceosome complexes using a reversible disulfide bond. This technique stabilizes RNA-protein interactions, aiding the study of spliceosome assembly dynamics.

Keywords:
Chemical modificationDisulfide crosslinkingNucleic acid–protein interactionsSpliceosomeU1 snRNP

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • The spliceosome is a large, dynamic molecular machine essential for gene expression.
  • Studying early spliceosome assembly is difficult due to its dynamic nature and transient complexes.

Purpose of the Study:

  • To develop a method for stabilizing non-covalent spliceosome complexes.
  • To enable biochemical and structural analysis of spliceosome assembly intermediates.

Main Methods:

  • Engineered a disulfide bond between a cysteine sidechain and a modified pre-mRNA.
  • Site-specifically modified pre-mRNA with an N-thioalkyl moiety.
  • Utilized oxidation to form a covalent crosslink, trapping complexes.

Main Results:

  • Successfully trapped an 11-subunit human U1 small nuclear ribonucleoprotein (snRNP) complex.
  • Demonstrated the ability to stabilize pre-mRNA/protein spliceosome complexes.
  • Provided protocols for applying this crosslinking strategy.

Conclusions:

  • This disulfide bond crosslinking approach effectively traps spliceosome complexes.
  • The method facilitates the study of dynamic RNA-protein interactions in spliceosome assembly.
  • Enables detailed structural and biochemical characterization of transient spliceosome intermediates.