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Title: Studies of proteins involved in pre-mRNA splicing
Author: Makarova, Olga
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1999
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Human SF2/ASF and hnRNP A1 are splicing factors that modulate alternative splicing in antagonistic manner: hnRNP A1 activates distal 5' splice sites and promotes alternative exon skipping, whereas SF2/ASF activates proximal 5' splice sites and promotes exon inclusion. The possibility that antagonism is a result of the specific RNA binding properties of these proteins was investigated in vitro using recombinant proteins and pre-mRNA. The binding of SF2/ASF and hnRNP A1 to RNA was compared by assays based on RNase H, nitrocellulose filter binding, and UV-crosslinking. HnRNP A1 dissociated from RNA within 5 seconds whereas the dissociation rate of SF2/ASF was much slower. The main determinant of hnRNP A1 binding was co-operativity but, in contrast, SF2/ASF may discriminate between different binding sites on the pre-mRNA. Close thermodynamic characteristics and very different kinetic parameters of binding were rationalised in the model that explains how the competitive binding of SF2/ASF and hnRNP A1 determines splice site recognition. Human Y chromosome-encoded RBM genes were identified on the basis of genetic analysis of infertile males. The structural features of RBM (RNA binding domain and SRGY-box repeats) evoked the possibility that it might function in splicing that was addressed here. Using antibodies, the expression of RBM was detected in human testis. For the first time, human U4/U6 snRNP particles were isolated that are able to associate with U5 snRNPs into [U4/U6.U5] tri-snRNP particles. A novel 61kD protein was identified as an integral component of the U4/U6 snRNP, and it appeared to be an orthologue of yeast splicing factor Prp31p. The U5-102kD protein, an orthologue of yeast Prp6p, was characterised, and it is likely to be involved in the interaction between U5 and U4/U6 particles through its multiple tetratrico peptide repeats.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available