Steps in the processing of Ad2 mRNA: poly(A)+ nuclear sequences are conserved and poly(A) addition precedes splicing

The conservation of nuclear Ad2 sequences during nucleocytoplasmic transport has been estimated from the accumulation of 3H-uridine in nuclear and cytoplasmic Ad2-specific RNA from the major late transcription unit. From 10-28% is conserved of the total Ad2 nuclear RNA synthesized from each of five regions of the genome that specify groups of 3' co-terminal mRNAs. The sum of the conservation of all the regions was equivalent to 100%, signifying the conservation of at least a part of each transcript or all of about one fifth to one sixth of the transcripts. The conservation of poly(A)+ Ad2 nuclear RNA is about 4 times greater than of total Ad2 nuclear RNA, approaching 100% conservation of poly(A)+ nuclear sequences. Since each mRNA contains three "spliced" sequences that are probably encoded only once per transcript, these data on conservation of the Ad2 sequences suggest that each transcriptional event from the 16-99 transcription unit gives rise to one of a possible 13-14 mRNA molecules with destruction of the remainder of the transcribed RNA. The portion which is conserved resides next to the region to which which poly(A) is added. Three models for the choice of poly(A) sites were considered: termination at the poly(A) site, cleavage shortly after synthesis of one of the sites before transcription was complete, and cleavage after completion of transcription. The first model was ruled out by the demonstration of equimolar synthesis over the 16-99 region. The second model is strongly supported because 3H-uridine label appears equally rapidly in the time range 2-10 min in each of the five 3' poly(A) addition sites, whereas chain completion before cleavage would lead to a faster appearance of label in the most promoter-distal site. Furthermore, briefly labeled RNA molecules extending from 16 to each of several poly(A) addition sites were the first poly(A)- terminated 3H-uridine-labeled molecules detected, demonstrating that poly(A) addition precedes splicing. The choice of which mRNA emerges from each transcriptional event would appear to depend upon first choosing one of five 3' mRNA ends followed by a 5' splicing event.