Báo cáo khóa học: Hydrolytic cleavage by a group I intron ribozyme is dependent on RNA structures not important for splicing pot

Hydrolytic cleavage by a group I intron ribozyme is dependenton RNA structures not important for splicing Peik Haugen1,*, Morten Andreassen2, A´sa B.. Results and discussion In this work

Hydrolytic cleavage by a group I intron ribozyme is dependent

on RNA structures not important for splicing

Peik Haugen1,*, Morten Andreassen2, A´sa B Birgisdottir

1

Department of Molecular Biotechnology, Institute of Medical Biology, University of Tromsø, Norway;2Faculty 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 essential for hydrolysis The most important positions in P9.2 include three adenosines in the terminal loop (L9.2) and

a consensus kink-turn motif in the proximal stem We sug-gest that the L9.2 adenosines and the kink-motif represent key regulatory elements in the splicing and hydrolytic reac-tion pathways

Keywords: Didymium iridis; group I intron; ribozyme hydrolysis; RNA processing; RNA structures

A highly conserved catalytic core is responsible for the

self-splicing reactions of group I intron ribozymes [1]

The secondary structures of paired segments (P1–P10 and

the optional P11–P17) are organized into three-dimensional

domains were P4–P6 and P3–P9 form the catalytic core [2,3]

The available crystal structure of the Tetrahymena intron

ribozyme core reveals an active site preorganized to catalysis

[3],which appears to contain at least three metal ions directly

involved in the reaction [4] The group I introns can be

divided into five main subgroups named IA,IB,IC,ID and

IE [2,5] The great majority of the more than 1200 group I

introns recognized within nuclear rDNA belong to the

group IC1 and group IE [6] While the Tetrahymena intron

(Tth.L1925) is a prototype group IC1 intron,the Didymium

twin-ribozyme intron Dir.S956-1 (and its DiGIR2

deri-vative) is the best studied of the group IE introns

Group I intron splicing is initiated by binding of an

exogenous guanosine (exoG) into the guanosine binding site

(GBS) Here,exoG is positioned for attack at the 5¢-splice

site (SS) and splicing proceeds through two consecutive

transesterification steps In addition to the essential exon

splicing reactions,Tth.L1925 also catalyze hydrolytic clea-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 andin 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 introduced by using the PCR based Quick Change site-directed mutagenesis kit (Stratagene) in combination with the following PAGE-purified oligonucleotides: pDiGIR2 L6,OP294/OP295; pDiGIR2 L9,OP 296/OP297; and pDiGIR2DP9.2

Correspondence to S Johansen,Department of Molecular

Biotechnology,Institute of Medical Biology,

University of Tromsø,N-9037 Tromsø,Norway.

Fax: + 47 77 645350,Tel.: + 47 77 645367,

E-mail: Steinar.Johansen@fagmed.uit.no

Abbreviations: GBS,guanosine binding site; HEG,homing

endonuclease gene; IGS,internal guide sequence; rDNA,

ribosomal DNA; SS,splice site.

*Present address: Department of Biological Sciences and Center

for Comparative Genomics,University of Iowa,Iowa City,

IA 52242–1324,USA.

(Received 30 October 2003,revised 24 December 2003,

accepted 19 January 2004)

constructs were made by cloning a PCR product into the

SmaI site of a pUC18 vector using the Sure Clone Ligation

Kit (Amersham Biosciences) PCR products were inserted

randomly into a forward of reverse position,according

to the M13 sequences flanking the multiple cloning site

The following primer combinations were used in order

to generate PCR products from the pDiGIR2

tem-plate Di347,OP129/OP347; Di348,OP129/OP348; Di349,

OP129/OP349; Di350,OP129/OP350; Inv310–318,OP129/

OP486; Inv312–314,OP129/OP487;

OP488; D

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

generate the pDi5DP9.2

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 http://www.fagmed.uit.no/

info/imb/amb

In vitro transcription, splicing and hydrolysis reactions

Precursor RNAs were transcribed from T7 promoters on

linearized plasmids in 25 lL

concentration (2 mM MgCl2) [35S]CTP[a

Amersham Biosciences) was uniformly incorporated into

the RNA transcripts The following plasmids were

linea-rized with EcoRI: pDi348,pDi349,pDi350,pDiGIR2,

pDiGIR2L6,pDiGIR2L9,pDiGIR2DP9.2

pDi5DP9.2

used in quantitative experiments were cut from

polyacryl-amide gels with surgical blades and incubated in 400 lL

elution buffer (0.3M NH4Ac,0.1% sodium dodecyl

sulfate,10 mM Tris pH 8 and 2.5 mMEDTA pH 8) on a

rotating wheel at 4C over night,purified through a

0.45 lM 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

to 30 lL of preheated (50C) buffer Reactions were

incubated under hydrolysis conditions (same as splicing

conditions,but without GTP) at 50C and samples

(5 lL) were collected,added to an equal volume of

STOP solution,and immediately frozen at )70C

Samples were separated on 8M 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 theIMAGEQUANT 3.3 software The 3¢ hydrolysis products were included as

a theoretical value Quantitative experiments involving impaired activity RNAs were performed once,while other experiments were reproduced between two and five times The hydrolysis data were fit to the nonlinear first-order decay equation with end-point correction

Ft¼ F4þ F0 ekobs xt previously described [15,16] Here, kobs is the calculated pseudo-first-order rate constant

Results and discussion

In this work we have used a full-length splicing construct (DiGIR2) and a 5¢-truncated DiGIR2 construct (Di347) in mutational studies to analyze hydrolytic cleavage at the 3¢-SS (Fig 1) Compared to DiGIR2,Di347 construct lacks the 5¢ exon,internal guide sequence (IGS),as well as the P1 and P2 elements

The 3¢ exon sequences are not essential for hydrolysis

at the 3¢-splice site Sequences flanking both the Tetrahymena and the Physarum introns [17,18] have been shown to influence

on the rate of in 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 8M 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 (kobs) are shown in Fig 2B below the plot Results indicate that the immediate 3¢-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 changes as in Di350 were introduced and tested in a DiGIR2 splicing context (DiGIR2.350) A time course experiment of DiGIR2.350 alongside the corresponding wild-type (DiGIR2.347) RNA is shown in Fig 2C The results indicate that the 3¢ exon sequence is not important for DiGIR2 splicing (see RNA 5),but some reductions in hydrolytic cleavage at the 3¢-SS (see RNA 3) and subsequent intron circle formation are observed (see RNA 1 and 6) This minor discrepancy between full-length splicing and 5¢-truncated transcripts may be due to RNA interaction of the proposed P10 (Fig 1B),which is present in the full-length splicing transcript but not the truncated transcript

Fig 1 Schematic presentation and secondary structure model of the DiGIR2 ribozyme from Didymium iridis (A) Schematic organization of the Dir.S956-1 intron (named according to [34]),encoding the two group I ribozyme motifs DiGIR1 and DiGIR2,and the I-DirI homing endo-nuclease The 5¢- and 3¢-splice sites (SS) are indicated,and flanking exon sequences are shown as open boxes Below: the DiGIR2 (lacking DiGIR1 and I-DirI ORF) and the 5¢-truncated DiGIR2 RNA (Di347) used in this study (B) Secondary structure model of DiGIR2 (modified from [7,8]) Intron RNA nucleotides and exon nucleotides are presented as upper case and lower case letters,respectively Paired segments (P) and intron nucleotides are numbered Long-range base pairing interactions (P10 and P13) are shown,and the first transcribed nucleotide of the 5¢-truncated Di347 RNA (position 40) is indicated by an arrow

GNRA to UUCG substitution of L9b, but not L6,

results in reduced 3¢-SS hydrolysis

GNRA tetra-loops have been shown to participate in

long-range RNA-RNA tertiary interactions in highly

structured RNAs,including the group I ribozymes,by

interacting with specific receptor structures [2,3,20]

GNRA tetra-loops may also function as local stabilizers

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 (kobs 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 spermidine,5 m M dithiothreitol,0.2 m M GTP] Exon nucleotides are presented

as lower case letters and substituted positions are shaded Pre,precursor RNA; Cut,5¢ RNA product; 3¢SS,3¢-splice site (B) Di347 and Di350 RNAs subjected to hydrolysis conditions (identical to splicing conditions but without GTP) and plotted as fraction uncleaved precursor (pre/total)

vs time Curves were fitted to the nonlinear first-order decay equation F t ¼ F 4 +F 0 · e)kobs xt and pseudo-first-order rate constants (k obs ) were calculated k obs variations represent differences between independent trials RNA bands were quantitated by phosphoimager exposure with IMAGEQUANT version 3.3 software (C) Self-splicing time course experiments (0–30 min) of DiGIR2.350 and wild-type DiGIR2.347 DiGIR2.347 was constructed in order to generate a DiGIR2 equivalent to DiGIR2.350 (short-3¢ exon sequences) Cir,intron RNA circle; Pre,precursor RNA; 5¢-E,5¢ exon; Int,Intron; LE,ligated exons.

rate (kobs 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),

construct,and analyzed for 3¢-SS hydrolysis The deletion

Fig 3 Analysis of DiGIR2 L6 and L9 GNRA tetra-loops in hydrolytic cleavage and self-splicing (A) Time course experiment (0–30 min) of 5¢-truncated variants containing tetra-loop substitutions,subjected to splicing conditions Di347L6 and Di347L9 have GUAA to UUCG and GCGA to UUCG substitutions in L6 and L9,respectively (B) Determination of hydrolytic cleavage rates,k obs ,at hydrolysis conditions of Di347L6 and Di347L9 (C) Self-splicing time course experiments (0–30 min) of the L6 (DiGIR2L6) and L9 (DiGIR2L9) substitution constructs See legends

to Fig 2 for abbreviations and experimental conditions.

had no effect on hydrolytic activity,consistent with that the

P5 internal loop could not serve as a GCGA L9b receptor

(data not shown)

The second context analyzed includes the second and

third base pairs in P5 stem,which harbors a potential

CU:AG (positions 97,98,128 and 129) receptor motif

(Fig 1B) This motif,at the exact same position in P5,is

frequently observed in combination with GNGA L9

tetra-loops in group IA introns [20] Studies have shown that

different GNRA tetra-loops appear to prefer a certain

receptor motif,but with a significant cross reaction [24,25]

Here,GUGA,GUAA and GAAA tetra-loops

preferen-tially interact with CU:AG,CC:GG and a 11 nt motif,

respectively To test for a possible P9b–P5 interaction,the

Di347 construct containing four different P9b GNRA

tetra-loop motifs (GCGA,GUGA,GUAA and GAAA),in

combination with two different putative P5 receptor motifs

at the second and third base pair positions (CU:AG and

CC:GG) were analyzed for hydrolytic cleavage

Further-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

the 5¢-truncated construct after  24 h of incubation (data

not shown) Surprisingly,the P9.2 deletion appears to

increase the splicing efficiency of DiGIR2 Figure 4B shows

that DiGIR2)P9.2 RNA was processed to essentially excised

intron and ligated exons Faint signals corresponding to

products of 3¢-SS hydrolytic cleavage (RNAs 3 and 7) and

intron circle formation (RNAs 1 and 6) are observed,

indicating that some hydrolytic activity are still present in

the full-length splicing construct compare to 5¢-truncated

construct

In order to evaluate the rate of hydrolytic cleavage

catalyzed by DiGIR2 and DiGIR2DP9.2

the Tetrahymena ribozyme (Tth.L1925) in a comparative

analysis RNA obtained from full-length splicing constructs

of the ribozymes was incubated under hydrolysis conditions (without GTP) The results are presented as an autoradio-gram (Fig 4C) and as a plot of fraction cleaved precursor

vs time (Fig 4D) DiGIR2 and Tth.L1925 were found to have very similar hydrolytic cleavage rates at their 3¢-SS at this reaction condition with a calculated kobsof 0.080 and 0.073 min)1,respectively The P9.2 deletion in DiGIR2 reduces the hydrolysis reaction approximately 10 fold (kobs

 0.007 min)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 (kobs less than 0.001 min)1; Fig 5B, C)

terminal loops within the P9 domain that are engaged in long-range base–pairing interactions,e.g P13 between L9.1 and L2.1,and P17 between L9 and L5 [26] Interestingly,

a putative base–pairing interaction exists in DiGIR2 between L9.2 (pos 312–314) and L5 (pos 114–116) However,experiments including the L9.2 mutant (Inv312– 314) excluded this possibility (kobs¼ 0.077 min)1 vs 0.085 min)1of wild type; Figs 5B,C) The remaining L9.2 positions were changed in pairs (310/311,315/316 and 317/318; Fig 5B),and the corresponding results are presented in Fig 5C All substitution mutants were found

to affect hydrolytic cleavage,with most dramatic effect at positions 315–318 (AAAU; kobs¼ 0.002–0.003 min)1) The impaired hydrolytic cleavage of the L9.2 substitu-tions is rescued by high Mg2+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 Mg2+ concentrations (5,10 and 50 mM) and

0 mMKCl A surprising observation was that the presence

of 200 mM K+(standard conditions) during the reaction has a negative effect on 3¢-SS hydrolysis Cleavage rates

Fig 4 Analysis of DiGIR2 P9.2 deletion in hydrolytic cleavage and self-splicing (A) Time course experiment (0–30 min) of Di347 and Di5)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

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 (D) Plot of fraction uncleaved precursor (pre/total) vs time of the hydrolysis reactions presented in (C),and subsequent determination of corresponding rate constants (k ) See legends to Fig 2 for abbreviations and experimental conditions.

24

were found to be significantly higher at 10 mMMg2+with

0 mMK+than 10 mMMg2+with 200 mMK+(compare Figs 5C and 6) Furthermore,the impaired hydrolytic cleavage in the mutant constructs is rescued by increasing the Mg2+concentrations,and at 50 mM all the mutants are at,or close to,the wild type level rate (Fig 6) Inhibition of Mg2+-dependent ribozymes by monovalent cations has previously been noted [27–29],and suggested

to be due to monovalent cations displacement of Mg2+ 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 (>3M) concentrations [29] Our observation that the impaired hydrolytic cleavage of the L9.2 substitutions is rescued by high Mg2+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

Fig 5 Analysis of DiGIR2 P9.2 substitutions and deletions in

hydro-lytic cleavage (A) Secondary structure presentations of P9.2 and the

P9.2 deletions (dashed lines) introduced into 5¢-truncated DiGIR2

ribozymes (B) Nucleotide substitutions introduced into the L9.2 loop

region of the 5¢-truncated DiGIR2 (C) Plot of fraction uncleaved

precursor (pre/total) vs time of the hydrolysis reactions presented in B

(hydrolysis conditions),and subsequent determination of

corres-ponding rate constants (k obs ) See legends to Fig 2 for abbreviations

and experimental conditions.

Fig 6 The effect of DiGIR2 L9.2 substitutions on hydrolysis by increased Mg 2+ concentration Time course experiment (0–150 min) of the wild type DiGIR2 and L9.2 substitution constructs schematically presented in Fig 5B at hydrolysis conditions with different magnesium concentrations,but at 0 m M KCl See legends to Fig 2 for abbrevia-tions and experimental condiabbrevia-tions.

Functional implications of P9.2 in hydrolysis

Our finding that P9.2 is an essential structural element in

hydrolytic cleavage,but does not affect splicing,contrasts

experimental data from the Tetrahymena group IC1 intron

P9.2 has been shown to be nonessential for splicing and

3¢-SS hydrolysis [30],but a deletion construct lacking both

P9.1 and P9.2 had a significant decrease in the folding rate

of the catalytic core [31] The detailed global structure of

TetrahymenaP9.2 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

the core [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

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

transition state interactions within the active site suggest

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

Regardless of the exact functional role in hydrolysis of the L9.2 nucleotides,hydrolysis is also dependent on proximal sequences in the P9.2 stem These sequences harbor a consensus kink (K)-turn motif (Fig 1B),fre-quently observed in ribosomal RNA and other structural RNAs as a protein binding motif [33] The K-turn motif

in P9.2 appears essential to DiGIR2 hydrolysis as deletion (D291–299/328–333)

hydro-lysis (Fig 5A) The K-motif in DiGIR2 probably both positions the essential L9.2 nucleotides for their functional role in 3¢-SS hydrolysis,and binds to a specific nuclear protein factor Recently we described that DiGIR2 processing proceeds in two independent reaction pathways (Fig 7),one leading to intron splicing and the other to full-length intron circularization via 3¢-SS hydrolysis [7] Here we suggest that the K-motif in P9.2 represents a key regulatory element between the reaction pathways,and that a protein factor may exists in the Didymium nucleus that participates in the regulation The observation that splicing appears much more efficient than hydrolysis

in vivo compared to in vitro [8,12–14] is consistent with this proposal

Acknowledgements

We thank Henrik Nielsen for discussions This work was founded by grants from Norwegian Research Council,Norwegian Cancer Society, Aakre Foundation for Cancer Research,and Simon Fougner Hart-manns Foundation.

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Fig 7.

25 Schematic representation of the functional implications for

hydrolysis in group I intron The splicing pathway (left) is initiated by

exogenous guanosine (exoG) and results in ligated exons and spliced

out free intron This pathway benefits the host organism The

circu-larization pathway (right) is initiated by hydrolysis at the 3¢-splice site

(SS),at the [ohgr]G residue (TG),and results in nonspliced exons and

full-length (FL) intron circle This pathway is a selfish feature for the

intron [7] The hydrolysis step is dependent on the P9-domain RNA

structures P9.2 and L9b,intron structures not important in the splicing

pathway.

I ribozymes with different functions in RNA splicing and

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