Insights into Branch Nucleophile Positioning and Activation from an Orthogonal Pre-mRNA Splicing System in Yeast

Duncan J. Smith, Maria M. Konarska, Charles C. Query

Research output: Contribution to journalArticle

29 Scopus citations

Abstract

The duplex formed between the branch site (BS) of a spliceosomal intron and its cognate sequence in U2 snRNA is important for spliceosome assembly and the first catalytic step of splicing. We describe the development of an orthogonal BS-U2 system in S. cerevisiae in which spliceosomes containing a grossly substituted second-copy U2 snRNA mediate the in vivo splicing of a single reporter transcript carrying a cognate substitution. Systematic use of this approach to investigate requirements for branching catalysis reveals considerable flexibility in the sequence of the BS-U2 duplex and its positioning relative to the catalytic center. Branching efficiency depends on the identity of the branch nucleotide, its position within the BS-U2 duplex, and its distance from U2/U6 helix Ia. These results provide insights into substrate selection during spliceosomal branching catalysis; additionally, this system provides a foundation and tool for future mechanistic splicing research.

Original languageEnglish (US)
Pages (from-to)333-343
Number of pages11
JournalMolecular Cell
Volume34
Issue number3
DOIs
StatePublished - May 15 2009

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Keywords

  • RNA

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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