Alterations in the pre-mRNA topology of the bovine growth hormone polyadenylation region decrease poly(A) site efficiency.

RNase mapping experiments show that the bovine growth hormone (bGH) poly(A) region forms an extensive hairpin loop. Mutants were prepared to change poly(A) region pre-mRNA structure and cleavage site efficiency without altering necessary sequences. An inverted repeat which includes the poly(A) cleavage site was created by insertion of a linker upstream of the poly(A) region to compete with any wild-type secondary structure. RNA mapping analyses show alterations in the nuclease accessibility of this mutant at the natural site of cleavage. This mutant shows a 75% drop in relative reporter gene expression at the steady-state protein and RNA levels. When the linker is inserted as a direct repeat, expression is equivalent to wildtype levels. To show that transcription was not terminated by the inverted repeat, the SV40 late poly(A) region was inserted downstream. These mutants show restored expression and processing at the downstream site. Our experiments reveal that the conformation of the poly(A) site pre-mRNA is important in mediating efficient cleavage-polyadenylation.