Year: 2015
Type: dissertation
Abstract: The spliceosomal E complex is the first spliceosomal complex that is committed\nto pre-mRNA splicing and therefore the components of this complex are early\ntargets for regulation. With the majority of splicing regulation occurring at these\nearly stages a greater understanding of these early complexes will bring a\ngreater understanding of how splicing is regulated. Even at this early stage the\nfunctional groups at either end of the intron are in close proximity but the\nproteins involved in bringing the splice sites together are still poorly\ncharacterised. This thesis studied three distinct threads: the first thread\ninvestigated the interactions of one of the proposed bridges between the two\nsplice sites, PRPF40A. Interaction studies in this thesis showed that the WW\ndomains of PRPF40A were able to precipitate the U2 snRNP. The FF domains\nof PRPF40A were also able to interact with another component of the E\ncomplex, the SMN protein. As yet it is unclear whether the interaction between\nPRPF40A and SMN is a direct interaction. What was striking in this study was\nthat no clear interaction with U1 snRNP could be seen with any of the fragments\nof the PRPF40A protein. This leads me to believe that, unlike yeast PRP40,\nPRPF40A is a U2 snRNP related protein, not a U1 snRNP protein. The second\nthread was to develop a new method to isolate U snRNPs using Flag-tagged U\nsnRNP proteins; in this study the U1 snRNP was purified using Flag-tagged\nU1A and a U1-U2 di-snRNP was isolated using Flag-tagged U2 snRNP. This\nmethod of epitope-tagging core U snRNP proteins is useful as an alternative to\nantibodies for isolation of specific U snRNPs from nuclear extracts, as it gives\nflexibility in the target protein. The final thread was the development of a method\nto isolate and analyse the composition of ATP independent complexes formed\non Exon and intron defined MINX constructs. The RNA composition was\nsuccessfully analysed in an isolated MINX intron defined complex and an\nisolated MINX exon defined complex. Both exon- and intron-defined MINX\nconstructs precipitated U1 and U2 snRNA. This study has focused on the\ninteractions of PRPF40A; it has confirmed the interaction of PRPF40A with U2\nsnRNP (Makarov et al., 2012) and shown that this interaction is through the\nWW domains of the protein. The study has also begun to elucidate the\ninteractions of the FF domains of the PRPF40A protein. Finally the study has\ndeveloped two methods for isolating and studying the formation of the\nspliceosomal E complex.
Author: Paul A. Smith
Language: English
Topic: RNA Research and Splicing
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W1427471595