Hydrolytic cleavage by a group I intron ribozyme is dependent
on RNA structures not important for splicing
Peik Haugen
1,
*, Morten Andreassen
2
,A
´
sa B. Birgisdottir
2
1
and Steinar Johansen
1,2
1
Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Norway;
2
Faculty of Fisheries and
Natural Sciences, Bodø Regional University, Norway
DiGIR2 is the group I splicing-ribozyme of the mobile twin-
ribozyme intron Dir.S956-1, present in Didymium nuclear
ribosomal DNA. DiGIR2 is responsible for intron excision,
exon ligation, 3¢-splice site hydrolysis, and full-length intron
RNA circle formation. We recently reported that DiGIR2
splicing (intron excision and exon ligation) competes with
hydrolysis and subsequent full-length intron circularization.
Here we present experimental evidence that hydrolysis at the
3¢-splice site in DiGIR2 is dependent on structural elements
within the P9 subdomain not involved in splicing. Whereas
the GCGA tetra-loop in P9b was found to be important in
hydrolytic cleavage, probably due to tertiary RNA–RNA
interactions, the P9.2 hairpin structure was found to be

vage at the 3¢-SS and the formation of truncated intron
circles [1,7]. Hydrolytic cleavage at the 3¢-SS is initiated
when the last intron nucleotide (TG) binds to the GBS prior
to exoG. Splicing and hydrolysis are competing reactions
leading to ligated exons and full-length intron circles,
respectively [7].
We have identified and examined an unusual category
of self-splicing group I introns with a complex structural
organization and function [8–11]. These twin-ribozyme
introns consist of two distinct ribozymes (GIR1 and GIR2)
and a homing endonuclease gene (HEG). The DiGIR2
ribozyme, encoded by the Didymium iridis twin-ribozyme
intron Dir.S956-1, catalyses intron splicing as well as a
pronounced 3¢-SS hydrolysis and subsequent intron circu-
larization in vitro as well as in vivo [7,8,12–14]. DiGIR2
represents the group IE introns, which has a different
structural organization than the Tetrahymena group IC1
intron. Here we report structural requirements of the 3¢-SS
hydrolysis reaction in DiGIR2, including the immediate
3¢-exon nucleotides, the P9.2 segment, and the GNRA tetra
loops in L6 and L9b.
Experimental procedures
Plasmid constructions and
in vitro
mutagenesis
3
pDiGIR2 containing the DiGIR2 ribozyme with flanking
exons is the basis for most constructs, and is previously
described [8]. The P9.2 stem deletion, as well as the L6 and
L9 GNRA to UUCG substitutions in DiGIR2, were

OP486; Inv312–314, OP129/OP487;
5
D300–327, OP129/
OP488; D
6
291–299/328–333, OP129/OP489; Inv317–318,
OP129/OP592; Inv315–316, OP129/OP593; Inv310–311,
OP129/OP594; DiGIR2347, OP39/OP347; DiGIR2350,
OP39/OP350. The OP129 and OP347 primer combination
was used to generate PCR products from the pDiGIR2 L6
and pDiGIR2 L9 templates, resulting in the pDi347 L6 and
pDi347 L9 constructs, respectively. OP129 and OP5 were
used to amplify a product from pDiGIR2DP9.2
7
and
generate the pDi5DP9.2
8
plasmid. Three different L9b
mutants were generated from the following primer combi-
nations. GUAA, OP559/OP560; GUGA, OP561/OP562;
GAAA, OP547/OP548. Mutants in the P5 region were
generated from the following primer combinations. CC-GG
P5 receptor, OP549/OP550; inverted P5 receptor, OP858/
OP859; P5 hinge, OP860/OP861. Oligonucleotide sequences
used in this work are available as supplement at the RNA
Research Groups web site at />info/imb/amb.
In vitro
transcription, splicing and hydrolysis reactions
Precursor RNAs were transcribed from T7 promoters on
linearized plasmids in 25 lL

sulfate, 10 m
M
Tris pH 8 and 2.5 m
M
EDTA pH 8) on a
rotating wheel at 4 °C over night, purified through a
0.45 l
M
single use filter (Millipore) with a 1 mL syringe,
and ethanol precipitated. RNA splicing was performed
under self-splicing conditions essentially as described [8].
Hydrolytic cleavage at the 3¢-splice site was started by
adding 15 lL of purified RNA (in DEPC
14
-treated water)
to 30 lLofpreheated(50°C) buffer. Reactions were
incubated under hydrolysis conditions (same as splicing
conditions, but without GTP) at 50°Candsamples
(5 lL) were collected, added to an equal volume of
STOP solution, and immediately frozen at )70°C.
Samples were separated on 8
M
urea/5% polyacrylamide
gels, followed by autoradiography.
Computations
To quantify RNA signals, phosphoimager screens were
scanned after one to several days of exposure and the
resulting images were analyzed by using the
IMAGEQUANT
3.3 software. The 3¢ hydrolysis products were included as

ontherateofin vitro splicing. To test for similar effects
of the 3¢ exon on DiGIR2 hydrolytic cleavage at the
3¢-SS, mutations were introduced into the eight first
positions of the 3¢-exon sequence (Fig. 2A) and analyzed
in both the 5¢-truncated and full-length splicing DiGIR2
contexts. Precursor (Pre) RNAs were incubated under
splicing conditions in time course experiments and the
generated RNA species were separated on 8
M
urea/5%
polyacrylamide gels. Compared to the wt exon context
(Di347; Fig. 2A), no reductions in hydrolytic cleavage of
truncated transcripts were observed even when 2–8 exon
positions were changed (Di348–50). Di347 and Di350
precursor RNAs were subjected to more extensive time
course experiments including quantification of radioactive
decay from the gels using phosphoimager screens.
Fraction hydrolyzed RNA (Cut) of the precursor was
plotted vs. time (Fig. 2B) and fitted into a nonlinear first-
order decay equation. The observed rate constants (k
obs
)
are shown in Fig. 2B below the plot. Results indicate
thattheimmediate3¢-exon sequence plays only a minor
role in DiGIR2 hydrolysis, which corroborates the recent
findings of the bacterial group IC3 ribozymes of Azoar-
cus and Synechococcus pre-tRNA [19]. Same mutational
changesasinDi350wereintroducedandtestedina
DiGIR2 splicing context (DiGIR2.350). A time course
experiment of DiGIR2.350 alongside the corresponding

of stem-loop structures analogous to the UNCG family
of tetra-loops [21]. UNCG tetra-loops have so far not
been found to participate in tertiary RNA–RNA inter-
actions. The DiGIR2 ribozyme contains two GNRA
tetra-loops; a GUAA in L6 and a GCGA in L9b
(Fig. 1B).
To evaluate the role of L6 and L9b in 3¢-SS hydrolytic
cleavage, the GNRA loops were replaced with UUCG, and
the corresponding constructs were analyzed in a similar
approach as described above. Results from time course
experiments involving the 5¢-truncated RNAs are shown in
Fig. 3A,B. A minor reduction in observed hydrolytic rate
(k
obs
0.085–0.077 min
)1
) was observed in the L6 UUCG
substitution construct (Di347L6) compared to that of the
wild type. However, the L9b UUCG replacement
(Di347L9) resulted in a 10 fold reduction of 3¢-SS hydrolysis
Fig. 2. Analysis of DiGIR2 3¢ exon sequences in hydrolytic cleavage and self-splicing. (A) Time course experiment (0–30 min) of 5¢-truncated
DiGIR2 containing different sequence substitution within the eight first positions of the 3¢ exon. Mutant RNAs were subjected to splicing
conditions [40 m
M
Tris pH 7.5, 10 m
M
MgCl, 200 m
M
KCl, 2 m
M

0.085–0.008 min
)1
). Time course experiments
of the corresponding full-length splicing constructs
(DiGIR2L6 and DiGIR2L9, respectively) and wild-type
DiGIR2 are shown in Fig. 3C. No significant difference
with respect to hydrolysis, circle formation, and exon
splicing could be observed between processed DiGIR2 and
DiGIR2L6 RNAs. We infer that the L6 GUAA tetra-loop
is not involved in RNA–RNA tertiary interactions. How-
ever, while the L9b substitution in DiGIR2 (DiGIR2L9)
does not affect splicing (RNA5), the amounts of hydrolysis
(RNA7) and intron circle formation (RNA1) are strongly
reduced. These observations are consistent with an RNA–
RNA tertiary interaction that involves the L9b GCGA
tetra-loop.
Search for an L9b tetra-loop receptor motif in P5
L9 GNRA tetra-loops, in combination with specific recep-
tors in P5, are common tertiary interactions within group I
introns [2,20,22,23]. Two different sequence contexts in P5
of DiGIR2 were tested for a possible receptor role with
GCGA L9b. The first sequence context analyzed was based
on the findings by Inoue and coworkers [23]. They reported
that the J5/5a hinge in the Pneumocystis group IC1 intron
may function as a receptor for the L9 GAAA tetra-loop.
The correspondent region in DiGIR2 appears to be the P5
internal loop (Fig. 1B). Thus, the internal loop was deleted
(D102–107, 121–124),
15
expressed as a DiGIR2 5¢-truncation

more, the CU:AG motif was inverted to a nonreceptor
sequence (GA:UC) and analyzed together with both the
wild-type GCGA and the GUGA P9b tetra-loops. Whereas
all the GNRA L9b tetra loops tested supported hydrolysis
well compared to UUCG, the wild-type tetra-loop (GCGA)
was always the most efficient one followed by GUGA.
However, no significant reduction in hydrolytic cleavage
rate with respect to wild type and mutant P5 constructs
could be found (data not shown). In summary, comparative
data support a P5 stem receptor [2,20,22], but we were not
able to gain further experimental evidence probably due to
a significant cross-reaction between the receptor motifs
used in our approach.
Deletion of P9.2 dramatically reduces 3¢-SS hydrolysis
The P9.2 paired segment is present in many nuclear
group IC1 and group IE introns, including the Tetrahym-
ena intron. However, no clear functional role has been
assigned to this peripheral structural element. To test a
possible functional importance in splicing and hydrolytic
cleavage, a deletion study of the P9.2 element was
performed. The first DiGIR2 deletion mutant to be
analyzed lacks the P9.2 structure (positions 293–331;
DiGIR2DP9.2)
16
. The corresponding 5¢-truncated and full-
length splicing constructs were transcribed and analyzed by
the same approach as described above. Time course
experiments are presented in Fig. 4, and revealed that the
P9.2 deletion dramatically reduces 3¢-SS hydrolysis
(Fig. 4A). In fact, no hydrolytic cleavage was detected in

)1
), which is similar to that observed in the P9b
tetra-loop substitution mutant (Fig. 3B).
Nucleotide positions within the L9.2 are essential
for hydrolysis
The observation that the P9.2 deletion dramatically affects
hydrolytic cleavage, but not splicing, suggests a more direct
role in ribozyme hydrolytic function. Two additional P9.2
deletion constructs were thus generated, and include a
proximal- (positions 291–299, 328–333) and a distal (posi-
tions 300–327) stem deletion (Fig. 5A). The corresponding
5¢-truncated constructs were transcribed and analyzed by
the same approach as described above, and found to
completely abolish the hydrolytic reaction (data not shown).
These results further support an important role of P9.2 in
3¢SS hydrolytic cleavage. We infer that the distal sequences
of P9.2 are essential as deletion of positions 300–327 did
not support hydrolysis. Furthermore, shortening of P9.2 by
the proximal deletion suggests a positional effect of the
distal sequences.
To test the importance of the P9.2 loop sequence (L9.2),
five different substitution mutants were generated in the
5¢-truncation constructs, in vitro transcribed, and subjected
to cleavage conditions. Indeed, L9.2 was found to be
essential for hydrolytic cleavage as substitution by inverting
all the L9.2 positions (positions 310–318; CGCUACAAA
to GCGATGTTT) became inactive (k
obs
less than
0.001 min

concentrations in the absence
of K
+
ions.
Further biochemical characterizations of the L9.2
mutants were performed, including hydrolysis at different
mono- and divalent cation concentrations. The corres-
ponding 5¢-truncation constructs were analyzed at three
different Mg
2+
concentrations (5, 10 and 50 m
M
)and
0m
M
KCl. A surprising observation was that the presence
of 200 m
M
K
+
(standard conditions) during the reaction
has a negative effect on 3¢-SS hydrolysis. Cleavage rates
1020 P. Haugen et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 4. Analysis of DiGIR2 P9.2 deletion in hydrolytic cleavage and self-splicing. (A)Timecourseexperiment(0–30min)ofDi347andDi5)P9.2
subjected to splicing conditions. (B) Self-splicing time course experiment (0–30 min) of DiGIR2 and DiGIR2DP9.2. The 3¢-SS hydrolysis and intron
circle
22;2322;23
formation are strongly reduced in DiGIR2DP9.2 compared to DiGIR2, while splicing appears unaffected. (C) Time course experiment of
DiGIR2, Tetrahymena ribozyme (Tth.L1925), and DiGIR2DP9.2, subjected to hydrolysis conditions (without GTP). DiGIR2DP9.2 was incubated
up to 21 h (1260 min) in order to complete the hydrolysis reaction for a more accurate calculation of the rate constant. M, RNA size marker.

concentrations, and at 50 m
M
all the mutants
are at, or close to, the wild type level rate (Fig. 6).
Inhibition of Mg
2+
-dependent ribozymes by monovalent
cations has previously been noted [27–29], and suggested
to be due to monovalent cations displacement of Mg
2+
from essential sites within the ribozymes [27]. Experiments
using the hammerhead ribozyme showed that instead of
having a coordinated stimulating effect on ribozyme
activity, Na
+
ions inhibit divalent ion mediated ribozyme
reactions at lower concentrations, while rescuing the
negative effect at higher (>3
M
) concentrations [29]. Our
observation that the impaired hydrolytic cleavage of the
L9.2 substitutions is rescued by high Mg
2+
concentrations
only in the absence of monovalent K
+
ions suggests that
magnesium plays an important role in hydrolysis, but is
being displaced (maybe from L9.2) in the presence of
monovalent ions.

Tetrahymena P9.2 is not known as peripheral extensions
outside the catalytic core were not included in the crystal
structure determination [3], but Fe(II)ÆEDTA cleavage data
and modeling indicate that P9.2 is pointing outwards from
thecore[26].
What is the functional role of the essential L9.2 nucleo-
tides in DiGIR2 hydrolysis? One possibility is that L9.2
nucleotides participate in tertiary contacts with other parts
of the molecule. Whereas all attempts to obtain supporting
indication or evidence of regular base-pairing interactions
have failed, the important L9.2 adenosines could still be
involved in, e.g. a minor helix packing interaction in an
unidentified, distally located receptor within DiGIR2
19
[32].
An alternative possibility is that P9.2 may serve a more
direct role in hydrolytic cleavage catalysis, perhaps by
presenting hydrolysis-dependent metal-ion (e.g. magnes-
ium) to the active site, as indicated by results presented in
Fig. 6. P9.2 could potentially access the catalytic core-region
during hydrolysis analogous to P1 during splicing. Experi-
mental data from the Tetrahymena intron have provided
strong evidence that the active site contains three magnes-
ium ions directly involved in catalysis [4]. The model for
transitionstateinteractionswithintheactivesitesuggest
that two of the metal ions are bound to the guanosine
cofactor and that the third metal ion interacts with a 3¢ atom
of the nucleotide preceding the intron. However, to our
knowledge none of the metal ions have been specifically
assigned to hydrolytic cleavage.

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