Studies of proteins involved in pre-mRNA splicing
thesisposted on 15.12.2014, 10:31 by Olga. Makarova
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.