Association of U2AF35, U2AF65 and U2 snRNP with constitutively and alternatively Spliced pre-mRNA

In the early stages of splicing, introns are bounded by splice sites at the 5’ and 3’ ends. Candidate 5’ splice sites are bound by U1 snRNPs (small nuclear Ribonucleoprotein). The 3’ splice sites comprise three distinct sequence elements. The branch point, the polypyrimidine tract and the 3’ splice site. These are bound by proteins, SF1 at the branch site and a heterodimer of UAF65 and U2AF35 that binds the polypyrimidine tract and 3’ splice site respectively. The U2 snRNP has been found associate loosely but in close proximity with the branch point in the early splicing complex E. The binding level of these proteins determines the usage of 3’ splice sites. At this stage, the pre-mRNA is committed to splice and splice sites are in close proximity to one another, but specific splice sites are not selected until complex A forms. It is known how candidate 5’ splice sites are selected, however, the mechanism of selection of candidate 3’ sites is still unknown. Here, using single molecule methods, it is shown that U2AF binds to pre-mRNA promiscuously until complex E has assembled; whereas U2 snRNP binds promiscuously until the formation of complex A. Complex A is formed by base pairing of a single U2 snRNP at the branch point region, this event is associated with the loss of U2AF proteins. With strong alternative sites, two copies of each factor bind, consistent with late selection. With two identical introns, the extent of splicing of the first intron U2 association controls the second intron U2 association. The defect in splicing of SMN2 exon7 is associated with poor binding of U2 snRNP rather than U2AF compared with SMN1 exon7. Moreover, some other splicing enhancer and silencer factors (such as Tra2β, RON ESE and PTB) could also affect the binding of U2AF and U2. Thus, as with U1 snRNPs, U2AF and U2 snRNP recognise candidate sites but their binding is not directly responsible for selection.