A 2-STEP MECHANISM FOR 5' AND 3' SPLICE-SITE PAIRING

A FUNDAMENTAL question in the splicing of precursor messenger RNA is h ow the 5' and 3' splice sites are recognized and paired during the spl icing reaction. It has been proposed that spliceosome assembly in meta zoan pre-mRNAs can be initiated through interaction between the 3' spl ice site and specific sequence elements on the downstream exon (an exo nic enhancer or a 5' splice site)(1). Pairing of the intronic 5' and 3 ' splice sites occurs subsequently. We report here that 5' and 3' spli ce sites located on separate synthetic pre-mRNA substrates can be effi ciently trans-spliced if the 3' trans-splicing substrate contains thes e downstream sequence elements. Moreover, selection of the trans 5' sp lice site can occur after the second pre-spliceosomal complex A has as sembled on the 3' trans-splicing substrate. Thus our data demonstrate that 5' and 3' splice-site pairing in metazoans can occur in two disti nct steps.