Use of Single Molecule Methods to Reveal the Mechanisms of Splice Site Selection

Understanding the molecular mechanisms that allow splice site selection is of fundamental importance to the study of alternative splicing. The study presented here has focused on the interactions of two spliceosomal trans-acting factors with pre-mRNA. The U1 snRNP is very important in 5’ splice site selection. However, how binding of U1 snRNP to pre-mRNAs containing multiple 5’ splice sites leads to 5’ splice site selection is unknown. To investigate the role of U1 snRNP in 5’splice site selection, single molecule microscopy was used to determine the number of U1 snRNPs bound to a single molecule of pre-mRNA. In cases where multiple strong 5’ splice sites are competing the furthest downstream 5’ss is used. In these cases, as many U1 snRNPs were observed bound to the pre-mRNA at an early stage of the reaction as there were 5’ splice sites, demonstrating that multiple sites were occupied. However, later pre-spliceosomal complexes contained only one U1 snRNP, indicating that the surplus U1 snRNP had been removed. A novel model of 5’ splice site selection is presented, in which U1snRNP stimulates the binding of SR proteins to the pre-mRNA and thus increases exon rigidity, enforcing use of the downstream 5’ splice site. The U2 Auxiliary Factor (U2AF), binds to the polypyrimidine tract and 3’ splice site of an intron and is a hetrodimer of the U2AF65 and U2AF35 subunits. The length of the polypyrimidine tract has been shown to influence the efficiency with which a 3’splice site is spliced. To investigate the role of polypyrimidine tract length on U2AF binding, experiments have been conducted in which the binding of U2AF65 to pre-mRNAs has been analysed using single molecule microscopy. Through the analysis of constructs derived from the Globin C pre-mRNA, the mechanisms of U2AF65 association with pre-mRNA have been shown to comprise; (i) a non-specific association that occurs with all RNA tested in the absence of ATP, (ii) a U1 snRNP-dependent association with pre-mRNAs in a polypyrimidine tract-independent manner, and (iii) an association with pre-mRNA in a polypyrimidine tract-dependent manner.