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Title: Arresting the spliceosome : investigations into the role of Snu114 within the spliceosome
Author: Harte, Steven
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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Splicing is the process where pre-mRNA is converted to mRNA via two transesterification reactions. With this process unwanted sequences of nucleic acids, known as introns, are removed allowing only the coding nucleic acid sequences, exons, to remain. This process is catalysed by a dynamically assembled, highly complex macromolecular machine called the spliceosome, which is made up of five small nuclear ribonucleoproteins (snRNPs). To date, the spliceosome has defied conventional methods for conclusive characterisation, resulting in it being relatively poorly understood, although advances have been made.1, 2 Apart from being of interest due to the fact that splicing is an essential life process, it is also of interest medically. Disruption to the splicing process can produce incorrectly formed mRNA, which plays a part in many diseases.3 Small molecule inhibitors which bind to, and inhibit, the functions of individual proteins would “stall” the spliceosome,4 circumventing its dynamic nature. These inhibitors could also form the basis of new drugs, treating diseases which incorrectly formed mRNA can cause. Previously reported small molecule inhibitors have inhibited splicing at the early stages of spliceosome assembly.5-7 However, our target protein snu1148 belongs to the U5 snRNP, which is involved later on in the splicing cycle. Inhibition of Snu114 should, therefore, lead to accumulation of spliceosome complexes produced at later stages of the cycle. Homology studies of Snu114 indicated a strong correlation of amino acid sequences with ribosomal growth factors EF-2 and EF-G. This study allowed us to target Snu114 using known EF-2 and EF-G inhibitors, sordarin and fusidic acid, which were tested and found to have significant splicing inhibition activity. A series of derivatives of these parent compounds were then attempted in an effort to improve splicing inhibition activity and to analyse the structure-activity relationship of fusidic acid and sordarin as splicing inhibitors. The biosynthesis of sordarin proved to be difficult and only a few derivatives were synthesised, however an improvement was made to splicing inhibition activity by forming sordaricin 32. Various fusidic acid derivatives were successfully synthesised, leading to an analysis of the structure-activity relationship of fusidic acid as a splicing inhibitor. Most fusidic acid derivatives produced a lower splicing inhibition activity than fusidic acid. However, fusidic acid derivative 229 had an equivalent inhibition activity to that found for fusidic acid. This result leads us to believe that the C-3 hydroxyl moiety of fusidic acid would be an ideal area for modification in future studies.
Supervisor: Berrisford, David Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Spliceosome ; Splicing ; Sordarin ; Fusidic Acid