Smith DJ, et al. (2009) Insights into branch nucleophile positioning and activation from an orthogonal pre-mRNA splicing system in yeast. Mol Cell 34(3):333-43
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.
|Status: Published||Type: Journal Article | Research Support, N.I.H., Extramural | Research Support, Non-U.S. Gov't||PubMed ID: 19450531|
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