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PRP18, a protein required for the second reaction in pre-mRNA splicing

Vijayraghavan, Usha and Abelson, John (1990) PRP18, a protein required for the second reaction in pre-mRNA splicing. Molecular and Cellular Biology, 10 (1). pp. 324-332. ISSN 0270-7306.

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We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

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Additional Information:Copyright © 1990 by the American Society for Microbiology. Recieved 25 July 1989 / Accepted 12 October 1989 We thank D. Horowitz and J. Banroques for their critical reading and comments on the manuscript. This work was supported by Public Health Service grant GM 32637 from the National Institutes of Health.
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Deposited On:25 May 2006
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