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Title: Molecular interactions of PRP8 protein in splicing complexes
Author: Whittaker, Erica
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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Nuclear pre-mRNA splicing occurs within the spliceosome. The spliceosome is a dynamic structure in which multiple interactions among splicing factors catalyze the removal of introns from pre-mRNAmolecules. The PRP8 gene of Saccharomyces cerevisiae encodes a 280 kD protein that is essential for splicing (Jackson et al., 1988). Previously, antibodies were raised against PRP8 protein, and an immunological approach was used to identify PRP8 protein as a stable component of both the U5 snRNP and the U4/U5/U6 multi-snRNP complex (Lossky et al., 1987). In this thesis, these immunological studies are extended, and the possible role of PRP8 protein as a spliceosomal pre-mRNA splicing factor is investigated. The association of PRP8 protein with yeast splicing complexes was demonstrated by the ability of PRP8-specific antibodies to precipitate precursor RNA, splicing reaction intermediates and lariat intron product from in vitro splicing reactions. PRP8 protein maintains its association with spliceosomal complexes throughout both steps of the splicing reaction and remains associated with the excised intron in a post-splicing complex that does not contain the spliced exons. The association of PRP8 protein with pre-mRNA molecules is splicing-dependent, for PRP8-specific antibodies did not precipitate mutant precursor RNAs nor did they precipitate wild-type pre-mRNA in the absence of ATP. A novel method for the immuno-affinity purification of yeast spliceosomes was developed. In vitro splicing reactions were assembled with polyadenylated pre-mRNA substrates. Poly(A)-binding protein (PAB), endogenous to yeast splicing extracts, associated with the poly(A) tail of the pre-mRNA, and spliceosomes were precipitated with antibodies raised against yeast PAB. Analysis of the affinity-selected spliceosomal proteins revealed the presence of PRP8 protein; thus is was established that PRP8 protein is a component of yeast spliceosomes. The possible function of PRP8 protein within spliceosomal complexes was explored by a combination of immunological and UV-crosslinking techniques. Short-wave UV light forms covalent crosslinks between adjacent protein and RNA molecules. PRP8-specific antibodies precipitated PRP8 protein-pre-mRNA adducts from UV-irradiated in vitro splicing reactions. This demonstrated that the two molecules were in direct contact at the time of exposure to UV light. The interaction between PRP8 protein and substrate RNA is a splicing-specific event. The protein-RNA interaction is dependent on the presence of ATP, and there was no crosslinking observed between PRP8 protein and RNA substrates that were not spliced, even if PRP8 protein assembled into complexes with these substrates. It was established that PRP8 protein would only interact with pre-mRNA molecules capable of participating in at least the first step of the splicing reaction, thereby implicating additional cis- or trans-acting factors in the PRP8 protein-pre-mRNA contact event. Finally, Ribonuclease T1 protection mapping studies were conducted to locate PRP8 protein's binding site(s) on pre-mRNA and pre-mRNA-derived molecules. Preliminary results are presented which suggest that PRP8 protein may interact with both the 5' splice site region of pre-mRNA molecules and the branch structure of the lariat intermediate and/or lariat intron. The implications of these results are explored.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available