A critical step in mRNA biogenesis is the generation of the mRNA 3′ end through an endonucleolytic cleavage of the primary transcript followed by the addition of a approximately 200 nucleotide (nt) poly(A) tail. The efficiency of poly(A) site function can vary widely and for those genes with multiple poly(A) sites, the choice can be a regulated event. A functional poly(A) site is characterized by cis‐acting RNA sequences including the well‐conserved AAUAAA hexamer, located 10–30 nt upstream of the cleavage site, and a highly variable downstream GU‐ or U‐rich element. The gene specific nature of the downstream sequence suggests that it may be a primary determinant of poly(A) site efficiency. Several recent studies have detailed the purification of factors that mediate the cleavage and polyadenylation reaction and that recognize the cis‐acting signals. Two of these factors are responsible for the formation of a stable, committed ternary complex with the pre‐RNA. In order to define the role of this stable complex in poly(A) site function, we have compared the processing efficiency of several pre‐mRNAs with the stability of the complex that forms on these RNAs. We show that ternary complex stability reflects both the in vivo and the in vitro efficiency of the poly(A) site and that the stability of this complex is dependent on the nature of the downstream sequence element. We conclude that the stability of these protein–RNA interactions, dictated by the downstream element, plays a major role in determining the processing efficiency of a particular poly(A) site.

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The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the Li poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the Li site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the Li poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the Li poly(A) site, contributes to the reduced use of the Li poly(A) site during the late stage of an adenovirus infection.The biogenesis of a eukaryotic mRNA is a complex process requiring the execution of a series of processing events to convert the initial RNA transcript into a mRNA. Included among these events is the processing of pre-mRNA to generate the mature 3' terminus of the mRNA (see reference 44 for a review). Although the majority of transcription units include only a single poly(A) site, there are examples of complex transcription units that utilize multiple poly(A) sites. Moreover, the choice of polyadenylation sites in complex transcriptional units can dictate the nature of the products and in some cases can be a regulated event (see reference 33 for a review). Perhaps the best such example is the regulated production of immunoglobulin heavy-chain mRNAs during B-lymphocyte maturation. Approximately equal amounts of the mRNAs encoding the secreted and membrane forms of the immunoglobulin (,u or -y) heavy-chain proteins are produced in the mature B cell, whereas in the terminally differentiated plasma cell, mRNA encoding the secreted form vastly predominates (see reference 25 for a review). Although it is likely that a change in poly(A) site use is a contributing factor (18,19,31,32,39), it is also true that alternative splicing contributes to this change (39,(48)(49)(50)).An additional example can be found in the adenovirus major late transcription unit, which produces five 3'-coterminal families of mRNA that encode the late viral proteins (see reference 45 for a review). The relative levels of the mRNA products vary during the course of virus infection, reflectin...