Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.652810
Title: Detection and analyses of proteins interacting with the U6 snRNA in SnRNP particles and in spliceosomes in the yeast Saccharomyces cerevisiae
Author: Incarnacion, V. V. P.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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Abstract:
The yeast U6 snRNA is the most highly conserved of the snRNAs involved in nuclear pre-mRNA splicing. U6 snRNA is a very important and dynamic molecule, undergoing several conformational changes during spliceosome assembly. In snRNP particles and in the spliceosome U6 snRNA is involved in numerous well characterised RNA-RNA interactions. In contrast, there have been few studies of the U6 snRNA-protein interactions. With the exception of the Prp24p splicing factor, no direct U6 snRNA-protein binding has been reported to date. An experimental procedure was developed to detect proteins associated with the U6 snRNA: In a yeast nuclear extract, functional U6 snRNP particles were reconstituted with a radiolabelled U6 snRNA produced by transcription in vitro. The extract was UV-irradiated to covalently crosslink proteins that are in close proximity to the U6 snRNA. Proteins were subsequently immunoprecipitated, either directly or via snRNP particles. After separation on SDS-PAGE, proteins UV-crosslinked to radiolabelled U6 snRNA were detected by autoradiography. Using this procedure, the splicing factors Prp24p, Uss1p and Prp8p were found to be in contact with the U6 snRNA. By blocking snRNP interactions and spliceosome formation at different stages we have shown that: Prp24p is in close proximity to U6 snRNA in the U6 snRNP; Uss1p is in contact with the U6 snRNA in the U6 and/or U4/U6 snRNP and also in the U4/U6.U5 tri-snRNP; Prp8p interacts with U6 snRNA in the tris-snRNP. Other (uncharacterized) proteins were also UV-crosslinked to the U6 snRNA. No contact between Prp2LAT mutant protein and U6 snRNA could be detected in spliceosomes.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.652810  DOI: Not available
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