Brr2 plays a role in spliceosomal activation in addition to U4/U6 unwinding

Nucleic Acids Res. 2015 Mar 31;43(6):3286-97. doi: 10.1093/nar/gkv062. Epub 2015 Feb 10.

Abstract

Brr2 is a DExD/H-box RNA helicase that is responsible for U4/U6 unwinding, a critical step in spliceosomal activation. Brr2 is a large protein (∼250 kD) that consists of an N-terminal domain (∼500 residues) with unknown function and two Hel308-like modules that are responsible for RNA unwinding. Here we demonstrate that removal of the entire N-terminal domain is lethal to Saccharomyces cerevisiae and deletion of the N-terminal 120 residues leads to splicing defects and severely impaired growth. This N-terminal truncation does not significantly affect Brr2's helicase activity. Brr2-Δ120 can be successfully assembled into the tri-snRNP (albeit at a lower level than the WT Brr2) and the spliceosomal B complex. However, the truncation significantly impairs spliceosomal activation, leading to a dramatic reduction of U5, U6 snRNAs and accumulation of U1 snRNA in the B(act) complex. The N-terminal domain of Brr2 does not seem to be directly involved in regulating U1/5'ss unwinding. Instead, the N-terminal domain seems to be critical for retaining U5 and U6 snRNPs during/after spliceosomal activation through its interaction with snRNAs and possibly other spliceosomal proteins, revealing a new role of Brr2 in spliceosomal activation in addition to U4/U6 unwinding.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Genes, Fungal
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Structure, Tertiary
  • RNA Helicases / chemistry
  • RNA Helicases / genetics
  • RNA Helicases / metabolism*
  • RNA Precursors / genetics
  • RNA Precursors / metabolism
  • RNA Splicing
  • RNA, Fungal / genetics
  • RNA, Fungal / metabolism
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism
  • Ribonucleoprotein, U4-U6 Small Nuclear / chemistry
  • Ribonucleoprotein, U4-U6 Small Nuclear / metabolism*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism*
  • Spliceosomes / metabolism*

Substances

  • Peptide Fragments
  • RNA Precursors
  • RNA, Fungal
  • RNA, Small Nuclear
  • Ribonucleoprotein, U4-U6 Small Nuclear
  • Saccharomyces cerevisiae Proteins
  • BRR2 protein, S cerevisiae
  • RNA Helicases