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accessory proteins (reviewed in
Rino and Carmo-Fonseca, 2009; Soller, 2006
).
Intron splicing is a process that is highly conserved among metazoans. Intron borders
and internal features are identified by short, partially degenerate consensus
sequences, to which individual components of the spliceosome have affinity.
Coordinated binding by multiple factors results in intron marking and subsequent
spliceosome formation. The vertebrate 5
0
splice site is identified by the consensus
sequence AG/GURAGU, while the 3
0
splice site is characterized by the consensus
YAG/N (
Soller, 2006; Wahl et al., 2009
)(
Fig. 1a
). An internal binding site, the
branch point, with a consensus sequence YURAY, is located somewhere within the
final third of the intron. Finally, a polypyrimidine tract extends 10-12 nt upstream
from the 3
0
splice site. While these splice site consensus sequences are conserved in
the fission yeast Schizosaccharomyces pombe, they are slightly different in the
budding yeast Saccharomyces cerevisiae (
Fig. 1b
). In this organism, the 5
0
splice
site consensus sequence is NN/GUAUGU, and the branch-point consensus
sequence is UACUAAC. There is usually no polypyrimidine tract, although the
sequence immediately upstream of the 3
0
YAG/N consensus splice site is often
pyrimidine-rich (
Kuhn and Kaufer, 2003
). In all organisms, introns containing
these splice-site consensus sequences are called GT-AG introns.
The splicing reaction happens in multiple steps: First, the U1 snRNP binds to the
5
0
splice site of the intron via base-pairing between the U1 snRNP sequence 3
0
-UC/
CAUUCA-5
0
and the 5
0
splice-site consensus 5
0
-AG/GURAGU-3
0
(
Aebi et al., 1987;
Seraphin et al., 1988
). The two subunits of the U2 snRNP auxillary factor (U2AF),
U2AF
65
and U2AF
35
, bind the polypryimidine tract and the AG nucleotides of the 3
0
splice site, respectively (
Merendino et al., 1999; Wu et al., 1999; Zorio and
Blumenthal, 1999a
). The SF1/BBP protein binds the branch point of the intron.
Next, the U2 snRNP can base pair with this same sequence, displacing SF1/BBP
from its binding site. Finally, the U1 and U2 snRNPs recruit the U4/U6
U5 tri-
snRNP, whose arrival stimulates additional rearrangements and the formation of the
catalytically active splicesosome (
Wahl et al., 2009
).
Splicing proceeds via two transesterification reactions. In the first step, the
spliceosome orients the intron so that the 2
0
hydroxyl of the branch-point adenosine
can attack the phosphodiester bond at the 5
0
splice site, producing an upstream exon
with a free 3
0
hydroxyl group and a branch-point lariat intron attached to the
downstream exon. In the next step of splicing, the spliceosome positions the free
Fig. 1
A comparison of the consensus sequences found in a typical (a) vertebrate intron with those
found in (b) S. cerevisiae and (c) C. elegans. The 5
0
and 3
0
splice sites are demarcated by slashes (/).
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