Splice site selection and ribonucleoprotein complex assembly during in vitro pre-mRNA splicing.

To study the determinants of splice site selection, we have inserted synthetic 5' and 3' splice sites at different positions within beta-globin genes and analyzed the resultant RNA substrates for in vitro splicing, factor binding, and complex assembly. We show that consensus 5' and 3' splice site sequences are insufficient to determine splice site utilization; in the presence or absence of the authentic site, the synthetic sites are variably active in a position-dependent manner. However, regardless of position or utilization, the synthetic 5' and 3' splice sites are bound by the appropriate splicing factors. Thus, binding of splicing factors is necessary but not sufficient for splice site utilization. Finally, we demonstrate that a block to efficient splicing can occur at multiple steps in the pathway of normal splicing complex assembly.