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Title: Studies of the proteins involved in the formation of early spliceosomal complexes
Authors: Smith, Paul
Supervisors: Makarova, Olga
Award date: 5-Jun-2015
Presented at: University of Leicester
Abstract: The spliceosomal E complex is the first spliceosomal complex that is committed to pre-mRNA splicing and therefore the components of this complex are early targets for regulation. With the majority of splicing regulation occurring at these early stages a greater understanding of these early complexes will bring a greater understanding of how splicing is regulated. Even at this early stage the functional groups at either end of the intron are in close proximity but the proteins involved in bringing the splice sites together are still poorly characterised. This thesis studied three distinct threads: the first thread investigated the interactions of one of the proposed bridges between the two splice sites, PRPF40A. Interaction studies in this thesis showed that the WW domains of PRPF40A were able to precipitate the U2 snRNP. The FF domains of PRPF40A were also able to interact with another component of the E complex, the SMN protein. As yet it is unclear whether the interaction between PRPF40A and SMN is a direct interaction. What was striking in this study was that no clear interaction with U1 snRNP could be seen with any of the fragments of the PRPF40A protein. This leads me to believe that, unlike yeast PRP40, PRPF40A is a U2 snRNP related protein, not a U1 snRNP protein. The second thread was to develop a new method to isolate U snRNPs using Flag-tagged U snRNP proteins; in this study the U1 snRNP was purified using Flag-tagged U1A and a U1-U2 di-snRNP was isolated using Flag-tagged U2 snRNP. This method of epitope-tagging core U snRNP proteins is useful as an alternative to antibodies for isolation of specific U snRNPs from nuclear extracts, as it gives flexibility in the target protein. The final thread was the development of a method to isolate and analyse the composition of ATP independent complexes formed on Exon and intron defined MINX constructs. The RNA composition was successfully analysed in an isolated MINX intron defined complex and an isolated MINX exon defined complex. Both exon- and intron-defined MINX constructs precipitated U1 and U2 snRNA. This study has focused on the interactions of PRPF40A; it has confirmed the interaction of PRPF40A with U2 snRNP (Makarov et al., 2012) and shown that this interaction is through the WW domains of the protein. The study has also begun to elucidate the interactions of the FF domains of the PRPF40A protein. Finally the study has developed two methods for isolating and studying the formation of the spliceosomal E complex.
Type: Thesis
Level: Doctoral
Qualification: PhD
Rights: Copyright © the author. All rights reserved.
Appears in Collections:Leicester Theses
Theses, Dept. of Biochemistry

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