Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.796803
Title: In vitro studies on mRNA 3' processing using a herpes simplex virus poly A site
Author: McWilliam, Robert
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 1992
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Abstract:
An in vitro mRNA 3' processing system was established. Nuclear extracts capable of supporting 3' processing in this system were prepared from both HeLa cells and K562 cells; this is the first report of the use of K562 nuclear extracts for in vitro 3' processing. While the processing activities of the K562 extracts appeared to be identical to those of HeLa extracts, their overall levels of activity were significantly lower. HeLa nuclear extracts were used to investigate the properties of a 67bp synthetic poly A site composed of sequences from the HSV-2 equivalent of the HSV-1 UL38 gene. It was established that the synthetic poly A site contained sequences necessary and sufficient to direct 3' processing in vitro in a sequence-specific manner. The efficiency of in vitro 3' processing at the synthetic poly A site was comparable to that obtained with the HSV-2 IE-5 gene poly A site. Experiments using variants of the synthetic poly A site, in which the spatial relationship of the essential sequence elements was altered, indicated that reducing or increasing the spacing between the poly A signal (AAUAAA) and the downstream, GU-rich sequence element caused a diminution of 3' processing activity. The quantitative and qualitative effects on 3' processing of the sequences between the poly A signal and the downstream sequence element were also investigated. Processing of a synthetic poly A site variant, in which the sequence between the poly A signal and the downstream sequence element were inverted, occurred at a different location from that normally observed and also with a slightly higher efficiency. This result indicated that the poly A signal and the downstream signal element are not solely responsible for defining the efficiency and accuracy of cleavage. Gel mobility retardation assays were used to investigate the interactions between nuclear processing factors and precursor RNAs derived from the synthetic poly A site and its variants. The synthetic poly A site formed the same processing-specific complexes as the HSV-2 IE-5 gene poly A site. The variants, in which processing efficiency was lower than that of the synthetic poly A site, did not efficiently form the processing-specific complexes, with one exception. In the latter case, the spacing between the poly A signal and the downstream sequence element was reduced to such an extent that binding of the complex components to the RNA was possible but processing was not. A transient CAT gene expression assay was used to analyse the efficiency of polyadenylation directed by the synthetic poly A site and certain of its variants in vivo. The relative CAT activities observed confirmed the results obtained using the in vitro 3' processing system. The interactions between processing factors and the synthetic poly A site were investigated using complementary RNAs and sense-strand DNA oligonucleotides. By annealing complementary RNAs to the precursor RNA prior to incubation with nuclear extract, it was shown that the presence of double-stranded RNA at either the poly A signal, the cleavage site or the downstream element prevented specific complex assembly and 3' processing. This indicates that processing factors interact with all three elements of the poly A site. Pre-incubation of nuclear extract with the oligonucleotide GGTGTGTT, which corresponds to the GU-rich downstream sequence, inhibited specific complex assembly and 3' processing. This suggested that this oligonucleotide interacted with a processing factor, perhaps that which binds to the downstream sequence element. The implications of such an interaction for 3' processing in vivo are discussed. Other DNA oligonucleotides, corresponding to the poly A signal, the cleavage site and the vector-derived 3' sequence, had no effect on 3' processing in vitro.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.796803  DOI: Not available
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