  Alternative cassette exons are known to originate from two processes
exonization of intronic sequences and exon shuffling. Herein, we suggest an
additional mechanism by which constitutively spliced exons become alternative
cassette exons during evolution. We compiled a dataset of orthologous exons
from human and mouse that are constitutively spliced in one species but
alternatively spliced in the other. Examination of these exons suggests that
the common ancestors were constitutively spliced. We show that relaxation of
the 59 splice site during evolution is one of the molecular mechanisms by which
exons shift from constitutive to alternative splicing. This shift is associated
with the fixation of exonic splicing regulatory sequences (ESRs) that are
essential for exon definition and control the inclusion level only after the
transition to alternative splicing. The effect of each ESR on splicing and the
combinatorial effects between two ESRs are conserved from fish to human. Our
results uncover an evolutionary pathway that increases transcriptome diversity
by shifting exons from constitutive to alternative splicing
