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Experimental Tests of Two Proofreading Mechanisms for 5=-Splice Site Selection Experimental Tests of Two Proofreading
Mechanisms for 5=-Splice Site Selection
Yangming Wang and Scott K. Silverman*
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801
A B S T R A C T Self-splicing group II intron RNAs catalyze a two-step process in
which the intron is excised as a lariat by two successive phosphodiester exchange
reactions. The reversibility of the rst step has been hypothesized to act as a
proofreading mechanism for improper 5=-splice site selection. However, without
synthetic access to mis-spliced RNAs, this hypothesis could not be tested. Here,
we used a deoxyribozyme to synthesize several branched RNAs that are derived
from the ai5 group II intron and mis-spliced at the 5=-splice site. Unlike the cor-
rectly spliced ai5 RNAs, the mis-spliced RNAs are observed not to undergo the
reverse of the rst step. This is well-controlled negative evidence against the
hypothesis that rst-step reversibility is a proofreading mechanism for 5=-splice
site selection. In a reaction equivalent either to the hydrolytic rst step of splicing
or to the hydrolytic reverse of the second step of splicing, a mis-spliced 5=-exon
can be trimmed to its proper length by the corresponding mis-spliced intron,
and in one case, the trimmed 5=-exon was observed to proceed correctly through
the second step of splicing. These ndings are the rst direct evidence that this
second proofreading mechanism can occur with a group II intron RNA that is mis-
spliced at the 5=-splice site. On the basis of the likely structural and evolutionary
relationship between group II introns and the spliceosome, we suggest that this
second proofreading mechanism may be operative in the spliceosome.
T
he RNA self-splicing pathway catalyzed by group II
intron RNAs (15) has two reaction steps
(Figure 1, panel a). In the rst step, an internal
adenosine 2=-hydroxyl group from domain 6 attacks the
5=-splice site phosphodiester linkage, forming a lariat
3=-exon intermediate with departure of the 5=-exon. In
the second step, the 5=-exon attacks the 3=-splice site,
forming the ligated exons and excising the intron as a
lariat. Although the rst step of splicing is reversible (6),
the underlying explanation for this reversibility is
unclear. The lack of natural 5=-splice site mis-splicing
(79) suggests that a proofreading mechanism may
exist. One specic hypothesis is that rst-step revers-
ibility is itself a proofreading mechanism: any intron that
mis-splices at the rst step by choosing an improper
5=-splice site can return to the initial unspliced state by
the reverse of the rst step (6). This would provide the
RNA another opportunity to splice correctly, rather than
waste the RNA molecule in a dead-end route or lead to
improperly spliced exons after subsequently proceeding
through the second step of splicing. This rst proof-
reading mechanism requires that mis-spliced introns
which have been formed by use of the improper
5=-splice site during the rst step will be competent to
proceed through the reverse of the rst step. However,
this mechanism has never been tested experimentally,
because the required mis-spliced RNAs could not be
synthesized using previously available methods.
Because a mis-spliced 5=-exon that proceeds through
the second step would lead to improperly ligated exons,
a natural mechanism to repair a mis-spliced 5=-exon
would be valuable. A second potential proofreading
mechanism for RNAs that are mis-spliced at the 5=-splice
site is for the incorrect 5=-exon to be converted to the
correct length 5=-exon and subsequently proceed
through the second step. When the incorrect 5=-exon
has one or more extra nucleotides due to mis-splicing
*To whom correspondence should
be addressed.
E-mail: scott@scs.uiuc.edu.
Received for review April 6, 2006
and accepted May 6, 2006.
Published online June 16, 2006
10.1021/cb6001569 CCC: $33.50
© 2006 by American Chemical Society
A
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ACS CHEMICAL BIOLOGY VOL.1 NO.5
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316 downstream of the correct 5=-splice site, this requires
trimming of the incorrect 5=-exon by removal of the
extra nucleotide(s), followed by the second step. Such
trimming of the 5=-exon could correspond to the well-
known hydrolytic rst step of splicing, in which water
instead of the branch-site adenosine acts as the nucleo-
phile that attacks the 5=-splice site (4, 1012). Alterna-
tively, the trimming reaction could instead correspond
to the hydrolytic reverse of the second step of splicing
(11, 13), analogous to spliced exons reopening (SER)
(1416). Hydrolysis has been observed for short RNA
oligonucleotides that are analogues of the 5=-exon (7, 8,
13). However, catalysis of oligonucleotide hydrolysis
and the subsequent second step have never been
observed by mis-spliced intron RNAs, because the
required mis-spliced RNAs could not be synthesized.
Therefore, a direct test of this second proofreading
mechanism for 5=-splice site selection has not been
achievable.
Our laboratory has identied many articial
deoxyribozymes (DNA enzymes) (17) for RNA liga-
tion (18, 19). Several of these deoxyribozymes
such as 7S11 (20, 21) create 2=,5=-branched RNA
(Figure 1, panel b) (2024), where a 2=,5=-branch is the
key structural element of the lariat RNAs that are the
intermediates in biological RNA splicing (25). In reac-
tions catalyzed by group II introns, branched RNA (which
lacks the closed loop of a lariat) is as functionally
competent as lariat RNA (4, 26). Here, we have used
synthetic branched RNAs prepared by the 7S11 deoxy-
ribozyme to allow explicit experimental tests of the two
proofreading mechanisms for 5=-splice site selection.
Mis-spliced RNAs are observed not to undergo the
reverse of the rst step of splicing, which provides
evidence against the rst proofreading mechanism.
In contrast, direct evidence is obtained for the opera-
tion of the second proofreading mechanism.
Figure 1. RNA splicing and branched RNA. a) The two steps of group II intron splicing. Interactions between the intron
binding sites (IBS) and exon binding sites (EBS) are also present during the rst step (not depicted). For the ai5 intron,
D123 is 675 nt and D56 is 77 nt; D4 is not depicted because it is dispensable for catalytic activity (52). First-step
reversibility has been observed for correctly spliced RNA (6); whether or not this reversibility applies for mis-spliced RNAs
is one focus of this manuscript. Hydrolytic cleavage at the 5 -splice site (4, 10 12) is also depicted (gray water molecule);
this leads to linear instead of branched RNA. b) Synthesis of 2=,5=-branched ai5 RNA catalyzed by the 7S11
deoxyribozyme (20, 21). For natural branched RNAs, the branch-site nucleotide is almost always adenosine (circled). For
applying 7S11 to synthesize branched RNAs that correspond to selection of an improper 5=-splice site, the DNA sequence
of paired region P4 (denoted here with Xs) is chosen to maintain base pairing with the RNA sequence near the 5=-splice
site. c) The key components of the branch-formation reaction, shown in greater atomic detail.
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VOL.1 NO.5 316324 2006
317 RESULTS AND DISCUSSION
Choice of the Splicing System. We used the ai5
group II intron RNA (27, 28) as the self-splicing RNA to
test the rst proofreading mechanism for 5=-splice site
selection. The ai5 RNA is a common model system for
understanding group II intron structure and function,
and it has been studied using several biochemical
approaches (4). One of the most useful approaches to
assemble ai5 is with the bipartite D123/D56 system
(Figure 2, panel a, shown sche-
matically after the rst step of
splicing; see boxed structure
for correctly spliced RNA). In
this bipartite system, the
branch-site adenosine nucleo-
tide of domain 6 is located
within the 77-nt D56 RNA that
comprises domains 5 and 6,
plus the 3=-exon (if any is
included; the rst splicing step
does not require the 3=-exon).
The 5=-exon is joined to the
5=-end of domains 13 (D123),
which encompasses 675 nt. A
key advantage of studying ai5
splicing is that a lariat topology
of D123/D56 is not necessary,
because both splicing steps
proceed well in the simpler
2=,5=-branched RNA (4, 26).
Synthesis of Mis-Spliced
ai5 Variants by the 7S11
Deoxyribozyme. Experimentally
testing the proofreading
mechanisms required
sy