Alternative use of a polyadenylation signal and of a downstream 3′ splice site: Effect of 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole

A minor pathway for the processing of transcripts from the early region 3 transcription unit of adenovirus 2 is described. It results from the selection of a promoter-proximal polyadenylation site P1 instead of the major promoter-distal sites P2 and P4. 5,6-Dichlorol-β-d-ribofuranosylbenzimidazole (DRB) considerably reduces the use of the major pathways and enhances the minor one. The characterization of three novel nuclear RNAs whose amount increases in DRB-treated cells leads us to propose the following steps for the selection of P1. The nascent transcript is first cleaved in the branch point-3′ splice site region of the first E3 IVS (intervening sequence), generating a promoter-proximal RNA with a heterogeneous 3′ end (F-RNA) and a promoter-distal RNA with a heterogeneous 5′ end (G-RNA). This cleavage prevents the formation of the premessenger RNAs ending at P2 and P4. The 3′ end of F-RNA is about 130 nucleotides downstream from P1 and F-RNA contains the signals required for cleavage-polyadenylation. It may thus generate CP1, which is the transcript ending at P1. The cleavage of the nascent transcript in a region crucial for spliceosome assembly suggests that defective assembly may render RNA sequences at the 3′ end of IVS accessible to intracellular nucleases and trigger the use of an upstream polyadenylation site. Such a mechanism may explain the alternative use of neighbouring, mutually exclusive, splice and polyadenylation sites.