Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing

@article{Moore1993EvidenceFT,
  title={Evidence for two active sites in the spliceosome provided by stereochemistry of pre-mRNA splicing},
  author={Melissa J. Moore and Phillip A. Sharp},
  journal={Nature},
  year={1993},
  volume={365},
  pages={364-368}
}
EXCISION of introns from nuclear precursors to messenger RNAs (pre-mRNAs) by the spliceosome requires two distinct phosphodiester transfer (transesterification) reactions: exchange of a 3′–5′ for a 2′–5′ bond in the first step (lariat formation) and exchange of one 3′–5′ phosphodiester for another in the second step (exon ligation)1–3. We report here determination of the stereochemical course of each step using splicing substrates that contained a chiral phosphorothioate. This has provided… 
Metal ion catalysis during splicing of premessenger RNA
TLDR
It is shown that in splicing reactions with a precursor RNA containing a 3′-sulphur substitution at the 5′ splice site, interaction between metal ion and leaving group is essential for catalysis of the first reaction step, establishing that the spliceosome is a metalloenzyme.
The two steps of group II intron self-splicing are mechanistically distinguishable.
TLDR
The results suggest that either two distinct active sites catalyze the two steps or that more significant alterations must be made in a single bifunctional active site to accommodate the two different reactions.
Stereochemical selectivity of group II intron splicing, reverse splicing, and hydrolysis reactions
TLDR
The stereochemical specificities of all of the major model hydrolytic reactions carried out by the aI5 gamma intron from Saccharomyces cerevisiae mitochondria are determined to clarify the relationships of these model reactions to the transesterification reactions of the intact self-splicing system and permit new studies to be interpreted more rigorously.
New tertiary constraints between the RNA components of active yeast spliceosomes: a photo-crosslinking study.
TLDR
New tertiary constraints between the RNA components are obtained and specific sites in U6, when substituted with a photoreactive 4-thiouridine or 5-iodouridine, produced spliceosome-dependent crosslinks to U2 snRNA, or in one case, to the pre-mRNA substrate.
Metal ion catalysis during the exon-ligation step of nuclear pre-mRNA splicing: extending the parallels between the spliceosome and group II introns.
TLDR
The results strengthen the argument that the spliceosome is an RNA catalyst that shares a common molecular ancestor with group II introns, and uses identical catalytic strategies for splicing.
Interactions between the terminal bases of mammalian introns are retained in inosine‐containing pre‐mRNAs.
TLDR
It is shown that complete substitution of guanosine residues by inosine in a pre‐mRNA has only a modest effect upon step 2 of splicing, although earlier spliceosome assembly steps are impaired.
Extensive interactions of PRP8 protein with the 5′ and 3′ splice sites during splicing suggest a role in stabilization of exon alignment by U5 snRNA.
TLDR
It is proposed that the large binding sites of PRP8 stabilize the intrinsically weaker interactions of U5 snRNA with both exons at the splice sites for exon alignment by the U5snRNP.
Metal ion catalysis during group II intron self-splicing: parallels with the spliceosome.
TLDR
It is shown that 3'-sulfur substitution at the 5' splice site of a group II intron causes a metal specificity switch during the first step of splicing, and striking parallels between the catalytic mechanisms employed by these two systems are provided.
Metal-ion coordination by U6 small nuclear RNA contributes to catalysis in the spliceosome
TLDR
Yeast U6 snRNA contributes to pre-messenger RNA splicing through metal-ion coordination, which is consistent with RNA catalysis by the spliceosome.
An RNA conformational change between the two chemical steps of group II self‐splicing.
TLDR
It is shown that the structural elements specific for the second splicing step are clustered in peripheral structures of domains II and VI, and this observations give further support to the existence of a single active site in group II introns.
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TLDR
The exon ligation reaction was successfully performed in reverse using unsubstituted intron RNA and ligated exons containing an Rp phosphorothioate at the exon junction site, suggesting that one non-bridging oxygen is particularly crucial for both splicing reactions.
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TLDR
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    Proceedings of the National Academy of Sciences of the United States of America
  • 1993
TLDR
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TLDR
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TLDR
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