Splice
site consensus sequences alone are insufficient to dictate the recognition of
real constitutive splice sites within the typically large transcripts of higher
eukaryotes, and large numbers of pseudo exons flanked by pseudo splice sites
with good matches to the consensus sequences can be easily designated. In an attempt to identify elements that
prevent pseudo exon splicing, we have systematically altered known splicing
signals as well as immediately adjacent flanking sequences of an arbitrarily
chosen pseudo exon from intron 1 of the human hprt gene. The substitution
of a 5' splice site that perfectly
matches the 5’ consensus combined with mutation to match the CAGG of the 3'
consensus failed to get this model pseudo exon included as the central exon in
a dhfr minigene context. Provision of a real 3' splice site and a
consensus 5' splice and the removal of an upstream inhibitory sequence was
necessary and sufficient to confer splicing on the pseudo exon. This activated context also supported the
splicing of a second pseudo exon sequence containing no apparent enhancer. Thus both the 5’ splice site sequence and
the polypyrimidine tract of the pseudo exon are defective despite their good
agreement with the consensus. On the
other hand, the pseudo exon body did not exert a negative influence on
splicing. The introduction into the
pseudo exon of a sequence selected for binding to ASF/SF2 or its substitution
with b-globin exon 2 only partially reversed the
effect of the upstream negative element and the defective polypyrimidine
tract. These results support the idea
that exon-bridging enhancers are not a prerequisite for constitutive exon
definition, and suggest that intrinsically defective splice sites and negative
elements may play important roles in distinguishing the real from the vast
number of false splicing signals.