A two-step mechanism for 5' and 3' splice-site pairing.

A fundamental question in the splicing of precursor messenger RNA is how the 5' and 3' splice sites are recognized and paired during the splicing reaction. It has been proposed that spliceosome assembly in metazoan pre-mRNAs can be initiated through interaction between the 3' splice site and specific sequence elements on the downstream exon (an exonic enhancer or a 5' splice site). Pairing of the intronic 5' and 3' splice sites occurs subsequently. We report here that 5' and 3' splice sites located on separate synthetic pre-mRNA substrates can be efficiently trans-spliced if the 3' trans-splicing substrate contains these downstream sequence elements. Moreover, selection of the trans 5' splice site can occur after the second pre-spliceosomal complex A has assembled on the 3' trans-splicing substrate. Thus our data demonstrate that 5' and 3' splice-site pairing in metazoans can occur in two distinct steps.