Abstract
Regulation of adenovirus major late transcription unit (MLTU) mRNA biosynthesis involves poly(A) site selection between five available sites, L1 through L5. The 5' proximal site completely dominates during early infection, whereas all five sites are used during late infection with L3 being favored slightly over the others. Previous studies have shown this early to late poly(A) switch will occur in the absence of MLTU-specific splicing patterns and hinges in large part on the character of the first poly(A) site. We have used in vitro assays to characterize basic features of the L1 and L3 pre-mRNAs which may help define how processing at poly(A) sites is controlled. We have found that L1 is 5-10-fold less efficient than L3 as a substrate for RNA cleavage. A primary difference between the L1 and L3 sites lies in the kinetics of their use, with cleavage at L3 occurring at twice the rate of cleavage at L1. In addition, L1 is 20-fold less effective than L3 in competing for processing factors. To investigate the sequence elements that contribute to poly(A) site efficiency, we have used competition assays in which the competitor RNAs lack upstream or downstream elements.
Highlights
Regulationofadenovirus major late transcription unit (MLTU) mRNAbiosynthesis involves poly(A) site selection between five available sites, L1 through L6
L1 is 6-10-fold less efficient than L3as a substrate for the poly(A) switch will occur independent of MLTU-specific
Deletion analyses of the L1 poly(A) site suggest that sequences 5‘ and 3‘ of the core AAUAAA play a role in allowing it to dominate over a downstream poly(A) site in early infections as well as in transfections [13, 14]
Summary
Regulationofadenovirus major late transcription unit (MLTU) mRNAbiosynthesis involves poly(A) site selection between five available sites, L1 through L6. This article must mRNA substrates involved and their interactionwith poly(A) site processing factors. I n Vitro Cleavage and Polyadenylation Assays-Unless stated otherwise, in vitro processing reactions were carried out at 30 "C for 30 min in the presence of 1 nM substrate RNA and 40% HeLa nuclear extract (2 mg/ml finalproteinconcentration).
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