Functional polyadenylation [poly(A)] sites consist of two sequence elements, the 
AAUAAA and G/U box signals, that closely flank the site of mRNA 3'-end 
formation. In agreement with previous results, random sequence insertions 
between the AAUAAA and G/U box signals were observed to inhibit poly(A) site 
function. However, sequence insertions of similar size that were predicted to 
form RNA stem-loop structures were found to have little effect on the efficiency 
of polyadenylation and instead induced a 3' shift in the site of polyadenylation 
that was equal to the length of the inserted stem-loop. The in vivo utilization 
of a poly(A) site bearing an internal RNA stem-loop structure was inhibited by 
mutations that destabilized the predicted stem but was restored by compensatory 
mutations. These results strongly support the hypothesis that the appropriate 
spacing of the AAUAAA and G/U box signals is critical for poly(A) site function. 
Sequence insertions that are able to form RNA secondary structures that maintain 
the correct spacing of these two RNA target sequences are well tolerated, 
whereas sequence insertions that disturb this spacing inhibit poly(A) site 
recognition. It is proposed that the effect of sequence insertions on poly(A) 
site function may be sufficiently predictable to allow the development of an 
assay for in vivo RNA secondary structure that uses poly(A) site selection as a 
readout.