Requirement of a downstream sequence for generation of a poly(A) addition site.

The 3' terminus of most, if not all, eucaryotic polyadenylated mRNAs is formed as a result of endonucleolytic cleavage of a larger precursor RNA. That is, transcription does not terminate at the mRNA 3' sequence but rather proceeds through this site, terminating at some distance downstream. Using a plasmid containing the adenovirus E2A transcriptional unit, we have investigated the sequence requirement for the formation of a mature mRNA 3' terminus, focusing on the role of sequences immediately distal to the poly(A) addition site. Deletion mutants were constructed in the region distal to the poly(A) addition site and assayed by transfection into human 293 cells. The results demonstrate that 35 nucleotides distal to the site of poly(A) addition are sufficient for the formation of a mature E2 mRNA. However, removal of an additional 15 nucleotides, leaving 20 nucleotides distal to the poly(A) site, abolished the ability to produce functional E2A mRNA. The defect in the production of functional mRNA from such a mutant appears to be in the proper cleavage of the primary transcript at the poly(A) addition site. It would thus appear that sequences immediately distal to the site of poly(A) addition do not contribute to the mature mRNA but are essential for the formation of mature mRNA.