POLYADENYLATION SITE SELECTION CANNOT OCCUR IN-VIVO AFTER EXCISION OFTHE 3'-TERMINAL INTRON

Splicing of 3'-terminal introns and polyadenylation of pre-mRNAs can b e coupled in an appropriate cell-free system. However, definitive evid ence has been lacking as to whether these events are coupled in vivo a nd whether the order of these two processing events is obligatory. Her e, we investigated these questions by examining the in vivo processing of transcripts that differ solely by the precise insertion of an intr on within the first of two polyadenylation signals. Quantitative S1 nu clease mapping and PCR techniques were utilized to analyze the process ed RNAs that accumulated in monkey cells transfected with plasmids enc oding these transcripts. We found that, whereas all of the primary tra nscripts that lacked the inserted intron were processed via utilizatio n of the 5'-proximal polyadenylation signal, none of the transcripts i nitially disrupted in this signal were processed this way even though the disrupting intron had been properly excised and excision sometimes preceded polyadenylation. In addition, deletion of the second polyade nylation signal resulted in failure of spliced transcripts to accumula te. We conclude that selection of, but not necessarily cleavage at the polyadenylation site precedes excision of the 3'-terminal intron in v ivo; although coupling exists during selection of the sites to be used for polyadenylation and excision of the 3'-terminal intron, the actua l order of the subsequent enzymatic reactions is probably simply a ref lection of their relative kinetics.