Regulated adenovirus mRNA 3'-end formation in a coupled in vitro transcription-processing system

The adenovirus major late transcription unit encodes five poly(A) sites whose use during infection is regulated. Early in the infection, the 5'-most site, L1, is used preferentially, whereas late in infection, all sites are used equivalently. Previous in vivo experiments indicated that regulatory sequences flank the AAUAAA and GU-rich elements of the L1 poly(A) site. We have developed an in vitro coupled transcription-processing system for studying the function of these regulatory sequences in HeLa cell nuclear extracts. The in vitro analysis using this system shows that predominant use of the L1 poly(A) site, as mediated by the upstream regulatory sequence, is independent of transcription. Furthermore, the reaction conditions are favorable to both 3'-end processing and splicing, making this system generally useful for the study of posttranscriptional processes.