Assembly and dynamics of the U4/U6 di-snRNP by single-molecule FRET

Nucleic Acids Res. 2015 Dec 15;43(22):10963-74. doi: 10.1093/nar/gkv1011. Epub 2015 Oct 25.


In large ribonucleoprotein machines, such as ribosomes and spliceosomes, RNA functions as an assembly scaffold as well as a critical catalytic component. Protein binding to the RNA scaffold can induce structural changes, which in turn modulate subsequent binding of other components. The spliceosomal U4/U6 di-snRNP contains extensively base paired U4 and U6 snRNAs, Snu13, Prp31, Prp3 and Prp4, seven Sm and seven LSm proteins. We have studied successive binding of all protein components to the snRNA duplex during di-snRNP assembly by electrophoretic mobility shift assay and accompanying conformational changes in the U4/U6 RNA 3-way junction by single-molecule FRET. Stems I and II of the duplex were found to co-axially stack in free RNA and function as a rigid scaffold during the entire assembly, but the U4 snRNA 5' stem-loop adopts alternative orientations each stabilized by Prp31 and Prp3/4 binding accounting for altered Prp3/4 binding affinities in presence of Prp31.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fluorescence Resonance Energy Transfer
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein-Serine-Threonine Kinases / metabolism
  • RNA Splicing Factors
  • Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
  • Ribonucleoprotein, U4-U6 Small Nuclear / metabolism*
  • Ribonucleoproteins, Small Nuclear / metabolism
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spliceosomes / metabolism


  • PRP31 protein, S cerevisiae
  • RNA Splicing Factors
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Ribonucleoproteins, Small Nuclear
  • Saccharomyces cerevisiae Proteins
  • Snu13 protein, S cerevisiae
  • PRP4 protein, S cerevisiae
  • Protein-Serine-Threonine Kinases