Ancient Alu elements have been shown to be included in mature transcripts by 
point mutations that improve their 5' or 3' splice sites. We have examined 
requirements for exonization of a younger, disease-associated AluYa5 in intron 
16 of the human ACE gene. A single G>C transversion in position -3 of the new 
Alu exon was insufficient for Alu exonization and a significant inclusion in 
mRNA was only observed when improving several potential splice donor sites in 
the presence of 3' CAG. Since complete Alu exonization was not achieved by 
optimizing traditional splicing signals, including the branch site, we tested 
whether auxiliary elements in AluYa5 were required for constitutive inclusion. 
Exonization was promoted by a SELEX-predicted heptamer in Alu consensus sequence 
222-228 and point mutations in highly conserved nucleotides of this heptamer 
decreased Alu inclusion. In addition, we show that Alu exonization was 
facilitated by a subset of serine/arginine-rich (SR) proteins through activation 
of the optimized 3' splice site. Finally, the haplotype- and allele-specific ACE 
minigenes generated similar splicing patterns in both ACE-expressing and 
non-expressing cells, suggesting that previously reported allelic association 
with plasma ACE activity and cardiovascular disease is not attributable to 
differential splicing of introns 16 and 17.