Báo cáo khoa học: Trans-splicing of a mutated glycosylasparaginase mRNA sequence by a group I ribozyme deficient in hydrolysis pptx

As an alternative to the Tetrahymena model ribozyme, the DiGIR2 group I ribozyme, derived from a mobile group I intron in rDNA of the myxomycete Didymium iridis, represents a new and att

Trans -splicing of a mutated glycosylasparaginase mRNA sequence

by a group I ribozyme deficient in hydrolysis

Eirik W Lundblad1, Peik Haugen1and Steinar D Johansen1,2

1

Department of Molecular Biotechnology, RNA Research group, Institute of Medical Biology, University of Tromsø, Norway;

2

Department of Fisheries and Natural Sciences, Bodø Regional University, Norway

RNA reprogramming represents a new concept in correcting

genetic defects at the RNA level However, for the technique

to be useful for therapy, the level of reprogramming must be

appropriate To improve the efficiency of group I

ribozyme-mediated RNA reprogramming, when using the

Tetrahy-menaribozyme, regions complementary to the target RNA

have previously been extended in length and accessible sites

in the target RNAs have been identified As an alternative to

the Tetrahymena model ribozyme, the DiGIR2 group I

ribozyme, derived from a mobile group I intron in rDNA

of the myxomycete Didymium iridis, represents a new and

attractive tool in RNA reprogramming We reported

recently that the deletion of a structural element within the

P9 domain of DiGIR2 turns off hydrolysis at the 3¢ splice site

(side reaction) without affecting self-splicing [Haugen, P., Andreassen, M., Birgisdottir, A˚.B & Johansen, S.D (2004) Eur J Biochem 271, 1015–1024] Here we analyze the potential of the modified ribozyme, deficient in hydrolysis at the 3¢ splice site, for application in group I ribozyme-medi-ated trans-splicing of RNA The improved ribozyme cata-lyses both cis-splicing and trans-splicing in vitro of a human glycosylasparaginasemRNA sequence with the same effi-ciency as the original DiGIR2 ribozyme, but without detectable levels of the unwanted hydrolysis

Keywords: glycosylasparaginase mRNA; group I intron; ribozyme hydrolysis; RNA reprogramming; trans-splicing

Group I ribozyme-mediated RNA reprogramming by

trans-splicing, has been successfully carried out using the

Tetrahymenaribozyme and various target RNAs [1–5] The

trans-splicing reaction is similar to the self-splicing reaction

normally catalysed by group I introns [6], except that the 5¢

exon is presented in trans and a corrected 3¢ exon is attached

to the ribozyme Ligation of these exons produces the

chimerical transcript that can be translated into a functional

protein RNA reprogramming is guided by a region

complementary to the target RNA (internal guide sequence,

IGS) located within the ribozyme, and the specificity and

efficiency of trans-splicing have mainly been improved by

extending the IGS [2,7–9] In addition, group I ribozymes

with randomised IGSs are used to identify regions on the

target RNAs that are accessible [1,4,10–13] In spite of

recent advances and significant efforts to optimize

trans-splicing reactions, the RNA reprogramming in cells remains

inefficient Moreover, group I ribozymes, including the Tetrahymenaribozyme, catalyze additional reactions that directly compete with splicing and probably lower the efficiency of trans-splicing Most pronounced is the 3¢ splice site hydrolysis of precursor RNAs [14–16], which is catalysed by the Tetrahymena ribozyme at a relatively high rate [16,17] Hydrolysis results in the formation of full-length intron RNA circles, which are commonly detected both in vitro and in vivo in a number of group I introns [17– 19] Designing ribozymes that catalyse little or no competing side reactions can therefore prove valuable in the search for better ribozyme tools that can be used in RNA reprogram-ming

DiGIR2 is the splicing ribozyme derived from the twin-ribozyme group I intron Dir.S956-1 in Didymium ribosomal DNA (Fig 1A) [20,21] DiGIR2 represents the group IE intron subgroup with clear distinction in structure compared to the distantly related Tetrahymena group IC1 intron [16,18,19] We recently reported that deletion of the P9.2 paired element in the DiGIR2 ribozyme (Fig 1B) significantly reduces hydrolytic clea-vage at the 3¢ splice site without affecting the self-splicing activity in cis-splicing constructs [16] The remarkable loss of unwanted side reactions, apparently without compromising splicing, identifies the new ribozyme con-struct (denoted DiGIR2DP9.2) as a potential improved tool in group I ribozyme-mediated trans-splicing of RNA Here we set out to investigate the ability and efficiency of DiGIR2 and DiGIR2DP9.2 to trans-splice RNA molecules Trans-splicing ribozymes were construc-ted and targeconstruc-ted against a mutaconstruc-ted glycosylasparaginase

Correspondence to S Johansen, Department of Molecular

Biotech-nology, Institute of Medical Biology, University of Tromsø, 9037

Tromsø, Norway Fax: + 47 77 64 53 50, Tel.: + 47 77 64 53 67,

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

Abbreviations: AGU, aspartylglycosaminuria; EGS, extended guide

sequence; GA, glycosylasparaginase; IGS, internal guide sequence;

nt, nucleotide; RPA, ribonuclease protection analysis.

Note: The oligonucleotide sequences used in this work are available on

request and as a supplement at the RNA Research Group’s website

at http://www.fagmed.uit.no/info/imb/amb

(Received 15 April 2004, revised 9 August 2004,

accepted 25 October 2004)

(GA) mRNA sequence Mutations in GA cause the

human lysosomal storage disease aspartylglycosaminuria

(AGU) [22]

Experimental procedures

Plasmid constructions andin vitro mutagenesis

The cis-splicing construct pDiGIR2 AGU was made by

combining two different PCR products The first product

contains the T7 promoter, 15 nucleotides (nt) from the

human GA open reading frame (ORF) and the DiGIR2

splicing ribozyme, and was generated from the pDiGIR2

template [20] using the primer combination OP340/341

The second product (108 nt) was amplified from a cloned

human GA cDNA template using the primer combination

OP342/346 The two PCR products were blunted,

phos-phorylated and ligated using the Sure Clone Ligation Kit

(Amersham Biosciences, Piscataway, NJ, USA) Finally, a

new PCR product was generated from the ligation mix

using the oligo primers OP341/346 and subsequently cloned into pUC18 The P9.2 hairpin was deleted from the pDiGIR2 AGU by using the Quick Change site-directed mutagenesis kit (Stratagene, Cedar Creek, TX, USA) and OP296/297, generating pDiGIR2DP9.2 AGU The trans-splicing constructs were made by PCR amplification with Pfu DNA polymerase (Promega, Madison, WI, USA) using pDiGIR2 AGU and pDiGIR2DP9.2 AGU as templates, generating the trans-splicing plasmid versions

of pDiGIR2 AGU and pDiGIR2DP9.2 AGU, respectively Here, the primer combinations OP1191/1192 and OP1191/

1202 were used The forward primer was designed with two sequences complementary to the target RNA [8 nt IGS and

9 nt extended guide sequence (EGS)] separated by a 5 nt wobble region The reverse primers were designed with alternative codons, of which the first 5 nt in the 3¢ exon are able to form a P10 helix with nucleotides in the IGS-wobble region The PCR products were digested with NotI and BamHI, gel extracted (QIAquick gel extraction kit; QIAGEN, Gmbh, Germany), and ligated downstream of

Fig 1 Constructs and structural features of

the DiGIR2 ribozyme (A) Organization of

the twin-ribozyme intron (Dir.S956-1) into

group I ribozyme motifs (DiGIR1 and

DiGIR2) and the I-DirI homing endonuclease

gene, as well as the two versions of the

DiGIR2 ribozyme used in this study The 5¢

and 3¢ splice sites (SS) are indicated, and

flanking exon sequences are shown as open

boxes (B) Secondary structure of the DiGIR2

ribozyme [16] Boxed nucleotides in P9.2 are

deleted in DiGIR2DP9.2 Intron RNA

nucle-otides and exon nuclenucle-otides are presented as

uppercase and lowercase letters, respectively.

the CMV- and T7-promoters into corresponding sites in a

pDNA3.1(–) vector (Invitrogen, Norway), which had the

nucleotide sequences between the NheI and XbaI sites

deleted to bring the inserts closer to the T7 RNA

polymerase transcription initiation site The target GA

RNA, containing the prevalent Finnish mutation (Fig 2A),

was PCR amplified with Pfu ultra HF DNA polymerase

(Stratagene) using the primer combination OP1219/1220

(containing NheI and BamHI sites, respectively) The PCR

product was digested with NheI and BamHI, gel extracted

(Qiagen gel extraction kit), and ligated downstream of the

CMV- and T7-promoters into corresponding sites in the

pDNA3.1(–) vector (Invitrogen) All constructs were

con-firmed by automatic sequencing by the ABI PRISM

BigDyeTerminator Cycle Sequencing Ready Reaction Kit

(PerkinElmer, Norway) running on an ABI Prism 377

system (PerkinElmer) Oligonucleotide sequences used in

this work are available on request or as a supplement at the

RNA Research Group’s website at http://www.fagmed

uit.no/info/imb/amb

In vitro transcription, splicing reactions and RT-PCR analysis

Precursor RNAs for cis-splicing analyses were transcribed from T7 promoters off BamHI-linearized pDiGIR2, pDiGIR2 AGU and pDiGIR2DP9.2 AGU plasmids [35S]CTP[aS] (10 lCiÆlL)1; Amersham Biosciences) was uniformly incorporated into the RNA transcripts RNA splicing was performed under self-splicing conditions essentially as described [20] Samples were separated on

8Murea/5% polyacrylamide gels, followed by autoradio-graphy To obtain the sufficient amounts of RNA the constructs were transcribed at 8 mM MgCl2, resulting in some splicing activity at time point 0 To analyse the ligated exon sequences from pDiGIR2 AGU and pDi-GIR2DP9.2 AGU RNAs, RNA corresponding to ligated exons was gel isolated and eluted in 400 lL of elution buffer (0.3M NH4Ac, 0.1% SDS, 10 mM Tris pH 8 and 2.5 mM EDTA pH 8) overnight on a rotary mixer at

4C RNA was subsequently filtered through a 0.45 lM

A

B

Fig 2 Cis-splicing experiments of DiGIR2-derived ribozymes inserted into GA RNA sequences (A) Top; schematic map of the human GA ORF indicating the intron inser-tion site at posiinser-tion 436 Mutainser-tions at posi-tions 488, 800 and 916 are frequently associated with the most common lysosomal degradation disorder AGU found in the Fin-nish, Spanish/American and American popu-lations, respectively [22] Middle; schematic drawing of RNA transcripts generated from constructs containing the DiGIR2 or DiGIR2DP9.2 ribozymes The ribozyme internal guide sequence (IGS) sequence was adapted to the heterologous exon sequence Bottom; similarities between flanking 5¢ and 3¢ exon sequences are noted between the Didymium rDNA and human GA ORF Underlined positions are identical (B) Left; time course cis-splicing experiment (0–30 min)

of DiGIR2 [in small subunit (SSU) rRNA] and the two GA ORF intron constructs DiGIR2 AGU and DiGIR2DP9.2 AGU The RNA species is present at time point 0 due to some splicing activity during transcription (Experimental procedures) Right; represen-tative result of a ligated exon sequence ladder obtained from an RT-PCR analysis of RNA

5 The DNA sequence is similar to the RNA sequence shown below The ligated exon junction is marked by an arrowhead.

single use filter (Millipore, Ireland), ethanol precipitated

and subjected to reverse transcription using the First

Strand Synthesis kit (Amersham Biosciences) and OP346

Ligated exons (120 bp) were amplified with OP346/421,

separated on a high percentage agarose gel, eluted using

the Agarose Gel Extraction kit (Boehringer Mannheim,

Mannheim, Germany), and finally cloned into pUC18

Two independent ligated exon cDNA clones from each

of the pDiGIR2 AGU and pDiGIR2DP9.2 AGU were

manually sequenced using the Thermo Sequenase

sequen-cing kit (Amersham Biosciences) and [33P]ddNTPs

(GATC; 450 lCiÆmL)1) as the label Precursor RNAs

for trans-splicing analyses (Fig 3) were in vitro

tran-scribed from T7 promoters off BamHI-linearized

plas-mids without [35S]CTP labeling The target GA RNA

transcript was 1050 nt A similar RT-PCR experiment,

as described above, was performed on the trans-spliced

products, but using the primer OP1194 in the RT

reaction and OP1193/1194 in amplification

Trans-splicing and ribonuclease protection analyses

In trans-splicing experiments, unnlabeled DiGIR2 AGU

or DiGIR2DP9.2 AGU RNAs and PAGE-purified GA

RNA were mixed in a 3 : 1 ratio Two microliters of 5·

low-salt buffer (40 mM Tris/HCl pH 7.5, 200 mM KCl,

2 mM spermidine, 5 mM dithiothreitol, 10 mM MgCl2, 0,2 mM GTP) was added and the volume was adjusted

to 10 lL with water The trans-splicing mix was incuba-ted at 37C for 3 h Ribonuclease protection analysis (RPA) was performed on 5 lL of trans-splicing RNA-mix by the RNase protection kit (Roche Applied Science, Penzberg, Germany) according to the manufacturer’s instructions The RPA probe was generated from the RT-PCR product of in vitro trans-spliced GA RNA (see above) cloned into the pGEM-T easy vector (Promega) This plasmid was linearized and transcribed from the SP6 promoter, labelling with [35S]CTP as described above, to get a RPA probe of larger size than the probe fragment protected by trans-spliced RNA in analysis by RPA The transcribed RPA probe was

 500 nt (Fig 4B) RPA samples were separated on 8M urea/5% polyacrylamide gels, followed by autoradio-graphy (Fig 3B) and phosphoimager quantitation (Fuji BAS 5000 system; IMAGE GAUGE 4.0 software) The cytosine content in the part of the RPA probe protected

by the different sized RNAs was calculated and included

as a theoretical value to make the intensities of different sized bands comparable The amount of reprogrammed product (RNA 2) was calculated as a fraction (in

A

B

Fig 3 Design of trans-splicing ribozyme

con-structs (A) The ribozyme contains the internal

guide sequence (IGS) and extended guide

sequence (EGS), which base-pairs to the

complementary sequence in GA mRNA

upstream of the mutation The ribozyme

catalyzes the coupled cleavage of mutated

mRNA and the ligation of the restorative 3¢

exon to the remaining 5¢ exon (B) The

ribo-zyme constructs used contain silent mutations

(underlined) introduced by alternative codons,

and a sequence tag used in RT-PCR detection

(boxed nucleotides) [4].

percentage) of reprogrammed product (RNA 2) + target

GA RNA (RNA 3) The amount of trans-splicing

ribozymes that had undergone the reaction [calculated

as the amount of reprogrammed product (RNA 2) as a

fraction (in percentage) of reprogrammed product (RNA 2) + trans-splicing ribozyme (RNA 4)] was similar for the two different ribozyme constructs (data not shown) Three parallels of RPA experiments were performed

Fig 4 Trans-splicing experiments of DiGIR2-derived ribozymes including the GA RNA as target sequence RT-PCR and ribonuclease protection analysis (RPA) of trans-spliced GA mRNA generated by DiGIR2 AGU and DiGIR2DP9.2 AGU ribozymes (A) Top; RT-PCR products from

in vitro trans-splicing analyses with DiGIR2 AGU and DiGIR2DP9.2 AGU corresponding to reprogrammed products of the expected size (361 nt) The negative control (Neg Ctrl.) contains first-strand synthesis master mix Below; representative result of trans-spliced GA RNA sequence obtained from RT-PCR The trans-splicing junction is marked by an arrow (B) Top; representative result of major RNA-species (numbered 1–4) detected in RPA RNA 1, undigested probe; RNA 2, trans-spliced GA mRNA; RNA 3, major GA band; RNA 4, major DiGIR2 AGU and DiGIR2DP9.2 AGU band Additional bands result from degradation of RNA during incubation at trans-splicing and RPA hybridization conditions Below; quantitation of RPA of trans-spliced GA mRNA generated by DiGIR2 AGU and DiGIR2DP9.2 AGU Comparative quantitative data were collected from three independent assays The splicing efficiency (percentage) was calculated by dividing the trans-splicing band (RNA 2) ·100 by the sum of the trans-splicing band (RNA 2) and the major GA band (RNA 3) As the amount of DiGIR2 AGU and DiGIR2DP9.2 AGU ribozymes added in the trans-splicing reactions was approximately identical, the addition of the ribozyme band (RNA 4) in the fractions denominator gave approximately identical comparative results (data not shown).

Results and Discussion

In vitro self-splicing of DiGIR2 and DiGIR2DP9.2

ribozymes from heterologous transcripts

To test the potential of DiGIR2 and DiGIR2DP9.2 in RNA

reprogramming, the ribozymes were first inserted in cis into

heterologous exons that represent therapeutic, relevant RNA

sequences The ribozymes were inserted between positions

436 and 437 of the human GA ORF sequence (Fig 2A) and

tested for splicing activity in vitro The most common

disorder of glycoprotein degradation, AGU, is caused by

mutations in GA [22] The nucleotides flanking the intron

insertion site in GA RNA, upstream of the prevalent Finnish

AGU mutation, is similar to the nucleotides flanking the wild

type Didymium rDNA insertion site (Fig 2A) In the

corresponding splicing constructs, the IGS was modified

from GGCCGCfiGGUCUU in order to adapt the

ribo-zymes to the GA 5¢ exon Figure 2B shows that the

IGS-modified DiGIR2 ribozyme excised itself from the precursor

RNA, and in the same process correctly ligated the

surrounding exons (DiGIR2 AGU, Fig 2B) Bands that

represent intron circle (RNA 1), precursor RNA (RNA 2), 5¢

exon–intron (RNA 3), free intron (RNA 4), ligated exons

(RNA 5), and free 3¢ exons (RNA 7), are visible The small

5¢ exon (RNA 6) was run off the gel The IGS-modified

DiGIR2DP9.2 AGU transcript generated a band pattern

analogous to DiGIR2 AGU, except for the

hydrolysis-dependent RNA species (RNAs 1, 3 and 7) that were absent

in the reaction (Fig 2B) In conclusion, these results show

that both the DiGIR2 and DiGIR2DP9.2 ribozymes

accu-rately self-splice when inserted into foreign exons in cis

In vitro trans-splicing of mutated GA mRNA sequences

using DiGIR2 and DiGIR2DP9.2 ribozymes

To test whether the DiGIR2 and DiGIR2DP9.2 ribozymes

are able to splice foreign exons also in trans, we targeted the

ribozymes to position 436 (uracil) in the mutated GA

mRNA (same site as in the cis-splicing experiment; Fig 3)

located upstream of the most common AGU mutations

(Fig 2) The ribozymes were designed with modifications

known to increase trans-splicing efficiency and specificity

[1,4,9]; an IGS of 8 nt was used, and based on work by

Sullenger and coworkers [5], a 9 nt EGS complementary to

the GA mRNA target was added (Fig 3) to further increase

specificity and efficiency Furthermore, a P10 helix of 5 nt

were included as this is shown to substantially increase

trans-splicing efficiency of the Tetrahymena ribozyme [9]

Finally, the 3¢ exon that contains the corrected AGU

sequence was degenerated by alternative codons (Fig 3) in

order to avoid strong intermolecular base-pairing to the

region complementary to the target RNA [4]

The trans-splicing ribozymes and RNA targets were

mixed and subjected to conditions that favour splicing (see

above) In an RT-PCR approach the trans-ligated exon

products were amplified and DNA sequenced to verify

correct splicing (Fig 4A) In order to quantify the amount

of trans-spliced product, and compare the trans-splicing

efficiency between DiGIR2 AGU and DiGIR2DP9.2 AGU,

we performed analysis by RPA The RPA probe was

designed to hybridize to a 312 nt region located upstream of

U436 in mutated GA mRNA and to a 49 nt region of the 3¢ exon in the ribozymes, resulting in a 361 nt protected region for the trans-spliced RNA The probe was in vitro transcribed containing additional vector sequences in order

to easily separate full-length probe from RNA fragments protected in analysis by RPA Gel analyses of RPA products (Fig 4B) confirmed the RT-PCR based experi-ment presented above of in vitro trans-splicing The amount

of trans-spliced products were approximately similar for DiGIR2AGU and DiGIR2DP9.2 AGU (1.8% and 2.0%, respectively)

In summary, the DiGIR2DP9.2 ribozyme deficient in hydrolysis is able to perform trans-splicing with high fidelity

in vitro at comparable rate compared to the wild-type derived DiGIR2 ribozyme The former ribozyme is smaller

in size and lacks the hydrolytic processing known to compete with intron splicing [18] Previous works on RNA reprogramming have focused on using the Tetrahymena ribozyme as the tool Our findings demonstrate that the DiGIR2 ribozyme (and its derivatives), in which 3¢ splice site hydrolysis can be assigned to defined structures within the intron [18], represent an interesting alternative to the Tetrahymenaribozyme Although the 3¢ splice site hydro-lysis side reaction is under control we realize that the challenge for the future will be to increase the fraction of reprogrammed mRNA Here, experiments that involve selection for better target accessibility and reprogramming [4,13,23] will be crucial

Acknowledgements

This work was funded by grants from The Norwegian Research Council, The Norwegian Cancer Society, The Aakre Foundation for Cancer Research, and Simon Fougner Hartmanns Foundation We thank Dr Ole K Tollersrud and Dr Hilde Monica Frostad Riise for the glycosylasparaginase cDNA plasmids.

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