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Title: Isolation and characterisation of a dominant negative mutant of the spliceosomal ATPase PRP2
Author: Plumpton, Mary
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1993
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Nuclear pre-mRNA splicing occurs within a multicomponent complex, the spliceosome, which is formed by the ordered assembly of small nuclear ribonucleoprotein particles (snRNPs), and a host of other protein factors. The PRP2 protein of Saccharomyces cerevisiae is a non-snRNP factor required for the first cleavage-ligation step of the nuclear pre-mRNA splicing reaction. It is not required for early stages of complex assembly, but associates transiently with spliceosomes prior to and throughout step 1. PRP2 is a member of the DEAD/DEAH box family of putative RNA helicases and has been shown to possess RNA-dependent ATPase activity. The highly transient association of PRP2 with spliceosomes has hindered biochemical studies of the interactions of this protein, and therefore a genetic approach, which has been used successfully to study protein-protein interactions in other systems, was adopted. The principle behind this approach is that mutations in the PRP2 gene that confer a dominant negative phenotype (ie. causing a dominant inhibitory effect over the wild-type) may do so by preventing release of PRP2 from splicing complexes. Stalled spliceosomes containing non-functional PRP2 would accumulate, facilitating genetic and biochemical analyses of this interaction. From a pool of randomly generated PRP2 mutants, one dominant negative allele, PRP2-dn1, was isolated that caused a decline in cell growth rate when overexpressed in a wild-type yeast strain. Cell growth inhibition was presumably the result of the observed defect in pre-mRNA splicing, and was partially alleviated by simultaneous co-overexpression of wild-type PRP2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available