Alternatively Spliced Genes
141
another DEXD-box containing RNA he-
licase together with other factors. The
release of the lariat intron and dissocia-
tion of U2, U5, and U6snRNPs from the
Icomp
lex
requires another putative RNA
helicase, hPrp43. The released U6snRNP
is reannealed with U4snRNP and associ-
ated with U5snRNP to form the U4/U6.U5
tri-snRNP complex to enter another cycle
of spliceosome assembly. The lariat intron
is debranched by the enzyme Dbr1. Many
spliceosomal components are presumably
recycled to form new spliceosomes.
It is clear that spliceosome assembly is a
highly dynamic process involving multiple
networks of RNA–RNA, protein–RNA,
and protein–protein interactions at each
step. Our understanding of this complex
process, as well as its regulation, remains
limited.
1.5
Biochemical Mechanisms of pre-mRNA
Splicing
The
complex
and
dynamic
process
of
spliceosome assembly culminates in the
formation of the catalytic core in which
5
0
ss and 3
0
ss are precisely juxtaposed.
The biochemical reactions of pre-mRNA
splicing
involve
two
transesteriFcation
steps (±ig. 4). The Frst step is the cleavage
at the 5
0
ss and the formation of the lariat
intermediate with a 2
0
-OH displacing a 3
0
-
OH group. The second step is the cleavage
at the 3
0
splice site with concomitant
ligation of the 5
0
and 3
0
exons in which
a3
0
-OH displaces another 3
0
-OH group.
Although it remains controversial, these
two steps of splicing reactions may take
place in two catalytic sites. In addition
to the chemical differences between the
Exon 1
P GU
AGU
G
A
A
Py
AG
Exon 2
P
5
ss
3
ss
Branch site
2
OH
P
U
G
A
OH
+
Step 2
3
splice site cleavage
and exon ligation
+
Step 1
P
Exon 1
Exon 2
OH
Exon 1
A
AG
P
U
G
Exon 2
P
5
splice site cleavage
and lariat formation
Fig. 4
The biochemical mechanisms of pre-mRNA splicing: two steps of
transesteriFcation. The phosphodiester linkages are indicated by the letter ‘‘p’’
inside a circle or a diamond.
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