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 language | English (US) |
---|---|
Pages (from-to) | 333-343 |
Number of pages | 11 |
Journal | Molecular Cell |
Volume | 34 |
Issue number | 3 |
DOIs | |
State | Published - May 15 2009 |
Keywords
- RNA
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology