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

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...

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Bibliographic Details
Published in:Molecular cell 2009-05, Vol.34 (3), p.333-343
Main Authors: Smith, Duncan J., Konarska, Maria M., Query, Charles C.
Format: Article
Language:English
Subjects:
Animals
Base Sequence
Humans
Introns
Molecular Sequence Data
Nucleic Acid Conformation
RNA
RNA Precursors - chemistry
RNA Precursors - genetics
RNA Precursors - metabolism
RNA Splicing
RNA, Small Nuclear - chemistry
RNA, Small Nuclear - genetics
RNA, Small Nuclear - metabolism
Saccharomyces cerevisiae - genetics
Saccharomyces cerevisiae - metabolism
Saccharomyces cerevisiae Proteins - genetics
Saccharomyces cerevisiae Proteins - metabolism
Sequence Alignment
Spliceosomes - genetics
Spliceosomes - metabolism
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Description
Summary: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.
ISSN:1097-2765
1097-4164
DOI:10.1016/j.molcel.2009.03.012