140
Alternatively Spliced Genes
heterodimer)
to
the
intronic
sequence
at the 3
0
splice site. Splicing reactions
can occur in the absence of detectable
U1snRNP. However, efFcient splicing of
most introns appears to require U1snRNP.
The formation of such an early complex
(
Ecomp
lex
) is considered to be the com-
mitment step that
directs the
nascent
pre-mRNA
transcript
into
the
splicing
pathway.
A distinct feature of the formation of
mammalian splicing commitment com-
plex is the involvement of a family of
proteins named SR proteins that are not
found in the genome of
S. cerevisiae
.SR
proteins share common structural fea-
tures, with one or two RNA recognition
motifs
(RRMs)
of
the
RNP-consensus
(RNP-cs) type at the amino-terminus and
a carboxyl domain rich in arginine and
serine
residues
(RS
domain,
Table 1).
These proteins play important roles in
recognizing exons and mediating inter-
actions between splice sites. SR proteins
interact with exonic sequences and re-
cruit splicing factors for the formation
of ‘‘cross-exon’’ complex, (i.e. exon def-
inition). SR proteins also interact with
5
0
ss and facilitate the ‘‘cross-intron’’ recog-
nition by mediating protein–protein in-
teractions between U1snRNP and factors
associated with the branch site 3
0
ss includ-
ing U2snRNP, U2A±, and possibly other
proteins.
The
binding
of
U1snRNP
and
SR
proteins
promotes
the
interaction
of
the
17S
U2snRNP
with
the
branch
site sequence. The stable association of
U2snRNP with the branch site to form
the
Ac
om
p
l
e
x
is
ATP-dependent
and
requires the U2snRNP-speciFc proteins
S±3a
[S±3a60/SAP61
(Prp9),
S±3a66/
SAP62
(Prp11)
and
S±3a120/SAP114
(Prp21)]. This process also requires non-
snRNP splicing factors including hPrp5
(a putative ATP-dependent RNA helicase).
Upon the integration of the U2snRNP
complex, a U2snRNP-protein, p14, con-
tacts the branch adenosine residue and
interacts with other U2snRNP proteins.
The next ATP-dependent step is the asso-
ciation of U4/U6.U5 tri-snRNP complex
to form the
B1 complex
,inw
h
i
c
ht
h
e
interaction of the 5
0
ss with U1snRNA
is destabilized by U5-100 kDa (hPrp28),
a putative RNA helicase. The 5
0
ss se-
quence is then engaged in interactions
with U6snRNA around the intronic region
and with U5snRNA at the exonic region.
After escorting the U6snRNP into the B1
complex, the U4snRNP is released to form
the
B2 complex
, a transition that requires
ATP and possibly U5-200 kDa (human ho-
molog of Brr2 in yeast, another putative
RNA helicase). The next ATP-dependent
transition is the formation of the
C1 com-
plex
, a process involving the activity of yet
another possible RNA helicase hPrp2 (hu-
man homolog of Prp2/Ynr011c). This may
directly lead to the formation of the cat-
alytic site for the
frst step oF splicing
,the
cleavage at the 5
0
ss with the formation of
a lariat intermediate (see ±ig. 4). U2, U6,
and U5snRNPs are associated with the
catalytically active form of splicing com-
plex in which the Frst step of splicing
occurs. The
second step oF splicing
(cleavage
at the 3
0
ss and ligation of exons) requires
an additional set of protein factors includ-
ing hPrp16, hPrp17, hPrp18, and hSlu7
(Table 1). The human homolog of the yeast
Prp16, hPrp16, contains an RS domain in
addition to DEXD-box helicase and AT-
Pase domains. The transition to form the
C2 complex
is again ATP-dependent. The
catalytic center for the second step is also
formed by U6snRNA, U2snRNA, and/or
associated proteins. The splicing products,
ligated exons, and the lariat intron, are
then released from the spliceosome by
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