Alternatively Spliced Genes
147
these ISSs contain extended polypyrimi-
dine tracts. Some splicing repressor ele-
ments contain sequences similar to au-
thentic splice sites, or decoy splice sites.
It has been proposed that such decoy
splice sites mediate nonproductive interac-
tions to suppress the usage of upstream 5
0
splice site. Again, several hnRNP proteins,
including
polypyrimidine
tract
binding
protein (PTB, also known as hnRNP I),
hnRNP A1, and hnRNP H play important
roles in splicing repression by ISSs.
One of the common features of these
cis-regulatory elements is that they are
not simple sequence elements that act
independently of each other. Sequence
elements have been identiFed that can
act to stimulate the splicing of one exon
but repress another exon (e.g. in ±G±R2
gene). Some regulatory elements contain
both enhancer and silencer domains. The
splicing of IgM exons M1 and M2 is
regulated by a sequence containing jux-
taposed splicing enhancer and silencer
elements. Alternative splicing of protein
4.1R pre-mRNA generates multiple iso-
forms. This alternative splicing event is
critical for red blood cell membrane bio-
genesis during erythroid differentiation
and is regulated by multiple cis-elements
and trans-acting regulators.
Splicing
regulatory
elements
usually
contain multiple binding sites for splicing
regulators
and
function
by
recruiting
other spliceosomal components to form
RNP-like
complexes.
An
evolutionarily
conserved
100
bp
intronic
suppressor
element in the caspase 2 (casp-2) gene,
In100,
speciFcally
inhibits
its
exon
9
splicing. Alternative splicing of this 61
bp exon 9 leads to the formation of two
functionally antagonistic products, casp-
2L and casp-2S. Casp-2L product promotes
cell death, whereas casp-2S prevents cell
death. The In100 element contains a decoy
3
0
splice site juxtaposed to a PTB-binding
domain, both of which contribute to the
full activity of In100 in inhibiting exon
9
inclusion.
The
upstream
portion
of
In100 contains a sequence with features
of an authentic 3
0
splice site (including
a
branch
site,
a
polypyrimidine
tract,
and AG dinucleotide). However, this site
is
not
used
under
normal
conditions.
This sequence is only recognized as a
3
0
s
swh
enth
es
i
t
ei
si
s
o
l
a
t
e
dw
i
thth
e
downstream PTB-binding domain deleted.
Biochemical and cell culture experiments
show
that
this
decoy
3
0
acceptor
site
interacts
nonproductively
with
the
5
0
splice site of the alternative exon 9, thus
repressing
the
efFcient
use
of
the
5
0
splice site of exon 9 despite a high level
of U1snRNP binding to this 5
0
splice
site. Downstream of the decoy 3
0
splice
site resides the second functional domain
that
interacts
with
PTB.
The
binding
of PTB to CU-rich motifs within this
downstream domain juxtaposed to the
decoy 3
0
acceptor site correlates well with
the repressor activity of this domain. PTB
can modulate recognition of the adjacent
decoy
3
0
acceptor
site.
In
addition
to
factors interacting with an authentic 3
0
splice site (such as U2A± or U2snRNP),
PTB as well as other proteins interact
with In100 and contribute to recognition
of
the
In100
decoy
3
0
splice
site
by
the spliceosome as an intronic repressor
element, rather than as an authentic 3
0
splice site. The regulatory role of PTB
in
casp-2
alternative
splicing
and
its
mechanism of action appear to be distinct
from other systems (Sect. 2.2.2). A recent
survey of known human genes involved
in
cell
death
regulation
suggests
that
In100-like intronic elements (i.e. 3
0
splice
site juxtaposed to PTB-binding domains)
may represent a general intronic splicing
repressor motif. Such intronic elements
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