The large N-terminal region of the Brr2 RNA helicase guides productive spliceosome activation

Genes Dev. 2015 Dec 15;29(24):2576-87. doi: 10.1101/gad.271528.115. Epub 2015 Dec 4.

Abstract

The Brr2 helicase provides the key remodeling activity for spliceosome catalytic activation, during which it disrupts the U4/U6 di-snRNP (small nuclear RNA protein), and its activity has to be tightly regulated. Brr2 exhibits an unusual architecture, including an ∼ 500-residue N-terminal region, whose functions and molecular mechanisms are presently unknown, followed by a tandem array of structurally similar helicase units (cassettes), only the first of which is catalytically active. Here, we show by crystal structure analysis of full-length Brr2 in complex with a regulatory Jab1/MPN domain of the Prp8 protein and by cross-linking/mass spectrometry of isolated Brr2 that the Brr2 N-terminal region encompasses two folded domains and adjacent linear elements that clamp and interconnect the helicase cassettes. Stepwise N-terminal truncations led to yeast growth and splicing defects, reduced Brr2 association with U4/U6•U5 tri-snRNPs, and increased ATP-dependent disruption of the tri-snRNP, yielding U4/U6 di-snRNP and U5 snRNP. Trends in the RNA-binding, ATPase, and helicase activities of the Brr2 truncation variants are fully rationalized by the crystal structure, demonstrating that the N-terminal region autoinhibits Brr2 via substrate competition and conformational clamping. Our results reveal molecular mechanisms that prevent premature and unproductive tri-snRNP disruption and suggest novel principles of Brr2-dependent splicing regulation.

Keywords: RNA helicase structure and function; X-ray crystallography; pre-mRNA splicing; remodeling of RNA–protein complexes; spliceosome catalytic activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Chaetomium / enzymology
  • Chaetomium / genetics
  • Crystallization
  • Humans
  • Models, Molecular*
  • Protein Binding
  • Protein Folding
  • Protein Splicing
  • Protein Structure, Quaternary
  • Protein Structure, Tertiary
  • RNA Helicases / chemistry*
  • RNA Helicases / genetics
  • RNA Helicases / metabolism*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism
  • Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
  • Ribonucleoprotein, U4-U6 Small Nuclear / metabolism
  • Ribonucleoprotein, U5 Small Nuclear / chemistry
  • Ribonucleoprotein, U5 Small Nuclear / metabolism
  • Ribonucleoproteins, Small Nuclear / chemistry*
  • Ribonucleoproteins, Small Nuclear / genetics
  • Ribonucleoproteins, Small Nuclear / metabolism*
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spliceosomes / enzymology*
  • Spliceosomes / genetics

Substances

  • PRP8 protein, S cerevisiae
  • PRPF8 protein, human
  • RNA-Binding Proteins
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Ribonucleoprotein, U5 Small Nuclear
  • Ribonucleoproteins, Small Nuclear
  • SNRNP200 protein, human
  • Saccharomyces cerevisiae Proteins
  • Adenosine Triphosphatases
  • BRR2 protein, S cerevisiae
  • RNA Helicases

Associated data

  • PDB/5DCA