138
Alternatively Spliced Genes
be assembled into the spliceosome. This
UsnRNP import process requires not only
general nuclear import factors such as
Importin-
β
but also speciFc factors such as
Snurportin-1 that recognizes the m3G cap
of the UsnRNP and interacts with general
factors for snRNP import into the nucleus.
Before
the
association
of
UsnRNP
particle-speciFc proteins, the UsnRNAs
undergo internal modiFcations including
pseudouridylation and 2
0
-O-methylation.
Such modiFcations appear to be necessary
for the assembly of a functional UsnRNP,
as shown for the human U2snRNP. These
posttranscriptional modiFcations are me-
diated by a small nucleolar (sno)RNA-
guided mechanism through the action
of snoRNPs. ±or example, U85 snoRNP
directs both 2
0
-O-methylation and pseu-
douridylation
of
U5snRNA.
±ollowing
these posttranscriptional internal modiF-
cations of snRNAs, the assembly of Us-
nRNPsiscomp
le
tedw
i
ththeassoc
ia
t
ion
with individual UsnRNP-speciFc proteins
(Table 1A). Among these UsnRNP-speciFc
proteins, U5-220 kDa (also named Prp8 or
PRPC8) is a crucial spliceosomal protein
that is most highly conserved through evo-
lution. Prp8 interacts with sequences of
all major splicing signals including 5
0
ss,
branch site, and 3
0
ss. It has been pro-
posed that Prp8 plays a critical role in
catalysis by aligning 5
0
ss, and 3
0
ss at the
catalytic center.
U6snRNP plays an important role at the
catalytic center of the spliceosome, and
its biogenesis has several unique features.
The U6snRNA is transcribed by RNA
polymerase III, and its cap structure is
a
γ
-monomethyl group. The U6snRNP
does not contain an Sm protein. Instead,
U6snRNP contains seven Sm-like proteins
(Lsm2, 3, 4, 5, 6, 7, and 8) interacting with
the U-rich region at the 3
0
end of the
U6snRNA. The assembly of U6snRNP is
believed to occur in the nucleus. The base-
pairing interactions between U4snRNA
and
U6snRNA
lead
to
the
formation
of the U4/U6snRNP, which associates
with
U5snRNP
as
the
U4/U6.U5
tri-
snRNP complex to join the spliceosome.
The formation of U4/U6.U5 tri-snRNP
complex and association of tri-snRNP with
the spliceosome require PRP±31 (human
homolog of yeast Prp31p) in addition to
other proteins.
1.4
Spliceosome Assembly
A large number of studies show that mam-
malian spliceosomes are assembled on the
pre-mRNA splicing substrate in an or-
derly fashion. A recent study, on the other
hand, reported a penta-snRNP particle pu-
riFed from the yeast extract. The Fnding
of this ‘‘preassembled’’ penta-snRNP par-
ticle suggests the potential importance of
concurrent multisite interactions during
spliceosome assembly among spliceoso-
mal components and different intronic
as well as exonic regions. Many com-
ponents of spliceosomes are conserved
between yeast and human. In fact, much
of our knowledge of spliceosome assem-
bly is based on genetic studies in yeast
and on biochemical experiments using
both yeast and mammalian systems. The
mammalian homologs of key spliceoso-
mal
components
have
been
identiFed
(Table 1). Systematic proteomic studies of
spliceosomes assembled on model splic-
ing substrates conFrm the high degree
of conservation between mammalian and
yeast spliceosomal proteins (Table 1).
Spliceosome assembly is a highly dy-
namic process, with multiple RNA–RNA,
RNA–protein, and protein–protein inter-
actions.
During
spliceosomal
assembly
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