Two regions downstream of AATAAA in the human antithrombin III gene are important for cleavage-polyadenylation.

We have investigated the sequence requirements for the cleavage-polyadenylation reaction in the human antithrombin III (ATIII) gene. A series of 5'-3' and 3'-5' deletions were produced around the AATAAA site using Bal31 nuclease. Ligation of appropriate pairs of such mutations resulted in the generation of varying sized deletions or duplications of sequences either upstream of, downstream of, or within the region encompassing the poly(A) site. Whereas a large deletion 3' to the AATAAA signal abolished cleavage and polyadenylation of ATIII transcripts, smaller deletions, all of which were subsets of the large one, did not. This indicated that the ATIII gene contains at least two independently acting poly(A)-cleavage signals 3' of AATAAA. When one of these signals was eliminated and the other was partially deleted at its 3'-end, we were able to disrupt the normal spacing between AATAAA and the cleavage site without substantially affecting the efficiency of the cleavage reaction. This suggested that the distance between AATAAA and the cleavage site is determined by the same sequence which, along with AATAAA, specifies cleavage and polyadenylation. The duplication of regions either upstream or downstream of AATAAA affected neither the efficiency nor the site of cleavage of the ATIII transcript. When a duplication included a large region containing AATAAA as well as downstream sequences, both sites were chosen for cleavage reactions. With a more delimited duplication, which included AATAAA but not the downstream cleavage signals, both sites were again used. However, two new cleavage sites were now detected. These results suggested that the distance between AATAAA sites and critical downstream cleavage-spacing sequences may also be important in determining the site of cleavage. This may in part stem from spatial constraints imposed by RNA-protein complexes which have been postulated to be critical in catalyzing the cleavage-polyadenylation reaction.