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Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA
Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA Anna Hitrik, Ghada Abboud-Jarrous, Natalie Orlovetskie, Raphael Serruya, Nayef Jarrous PII: DOI: Reference:
S1874-9399(16)30001-3 doi: 10.1016/j.bbagrm.2016.01.004 BBAGRM 981
To appear in:
BBA - Gene Regulatory Mechanisms
Received date: Revised date: Accepted date:
24 August 2015 29 December 2015 21 January 2016
Please cite this article as: Anna Hitrik, Ghada Abboud-Jarrous, Natalie Orlovetskie, Raphael Serruya, Nayef Jarrous, Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA, BBA - Gene Regulatory Mechanisms (2016), doi: 10.1016/j.bbagrm.2016.01.004
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Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA
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Anna Hitrik1, Ghada Abboud-Jarrous2, Natalie Orlovetskie1, Raphael Serruya1, and Nayef
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Department of Microbiology and Molecular Genetics, The Hebrew University-Hadassah
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Medical School, Jerusalem 91120, Israel 2
Institute for Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine,
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Jerusalem 91120, Israel
Corresponding author: Tel.: 972-2-6758233; Fax.: 972-2-6784010; e-mail:
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Jarrous1,2
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[email protected]
Running title: EGS-directed knockdown of WRN helicase
Keywords: External guide sequence; RNase P RNA; WRN helicase, selective cancer therapy; Werner Syndrome
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ACCEPTED MANUSCRIPT Abstract Human WRN, a RecQ helicase encoded by the Werner syndrome gene, is implicated in
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genome maintenance, including replication, recombination, excision repair and DNA damage
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response. These genetic processes and expression of WRN are concomitantly upregulated in
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many types of cancers. Therefore, targeted destruction of this helicase could be useful for elimination of cancer cells. Here, we provide a proof of concept for applying the external
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guide sequence (EGS) approach in directing an RNase P RNA to efficiently cleave the WRN mRNA in cultured human cell lines, thus abolishing translation and activity of this distinctive
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3’-5’ DNA helicase-nuclease. Remarkably, EGS-directed knockdown of WRN leads to inhibition of cell viability and proliferation. Hence, further assessment of this targeting
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system could be beneficial for selective cancer therapies, particularly in the light of the
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recent improvements introduced into EGSs.
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ACCEPTED MANUSCRIPT Introduction Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging
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and increased frequency of cancer [1-4]. WS belongs to a family of progeroid syndromes,
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which include Hutchinson–Gilford progeria syndrome, Cockayne syndrome, ataxia-
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telangiectasia and Down syndrome [5,6] and is caused by mutations in the Werner Syndrome gene, WRN, which codes for a conserved RecQ helicase [1,5]. Human WRN
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protein consists of several domains that coordinate enzymatic activities, such as 3’-5’ DNA helicase and exonuclease activities [7,9], which are implicated in DNA maintenance and
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telomere preservation [1,6-10]. These enzymatic activities assist cells in removing unresolved replication intermediates and renewal of DNA replication [7]. Forked dsDNA and
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bubble DNA structures are preferred substrates for WRN [7,11], which facilitates a helicase-
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enhanced strand displacement in coordination with DNA Pol β [4,7]. WRN is also involved in
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DNA damage response via the activation of the poly(ADP-ribose) polymerase 1 [4,12-15]. These roles may explain why WS patients are predisposed to genomic instability and cancer
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[4,16]. Surprisingly, several studies showed that expression of WRN is elevated in many types of cancers, which are unrelated to those described in WS patients [4,17,18], possibly to stimulate replication progression and cell proliferation. Thus, this helicase may constitute a potential target for selective cancer therapy [4,18-20]. It has been shown that inhibition of WRN in cancer cells by small interfering RNAs (siRNAs) or small-molecule inhibitors leads to cell death via attenuation of the repair of replicative DNA defects and weakening of the DNA damage response [18-21]. Moreover, siRNA knockdown of WRN or its related RecQL1 helicase improves the chemotherapeutic efficacy of the topoisomerase inhibitor camptothecin against HeLa cells [20,23,24].
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ACCEPTED MANUSCRIPT Nonetheless, the utilization of siRNA has general limitations, such as off-target effects and instability [19,25].
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The external guide sequence (EGS)/RNase P system constitutes a powerful targeting
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approach for inhibition of gene expression [26-28]. An EGS is a short RNA designed to anneal
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with a target RNA, thus forming a hybrid RNA with a precursor tRNA-like structure that is specifically recognized and cleaved by RNase P [26-28]. RNase P is a ubiquitous
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ribonucleoprotein endoribonuclease that processes the 5' leader of precursor tRNA through recognition of structural features [26,27,29,30]. The EGS/RNase P system was shown to
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destroy various types of RNAs, pathogenic or otherwise, in cells and in model organisms [3137]. Moreover, the catalytic M1 RNA subunit of Escherichia coli RNase P, which possesses C5
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protein cofactor, functions in EGS-directed targeting of gene expression in mammalian cells
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[27,28], even though the molecular mechanism of its action is unknown.
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In this study, we report on the design and construction of an EGS RNA that guides M1 RNA to efficiently knock down WRN at the mRNA, protein and activity levels in cultured
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human cell lines. Knockdown of this DNA helicase leads to inhibition of viability of cell lines. Therefore, this targeting system constitutes a potential tool for selective cancer therapy in the future.
Materials and Methods Gene constructs A 152-bp WRN cDNA fragment, which spanned the translation initiation region, was prepared by digesting of a full length WRN cDNA [1] with Kpn I and Bam H1. The released cDNA fragment was then gel-purified and cloned in pBluescript(SK) linearized by the same restriction enzymes (Fig. S1A). The sense WRN mRNA strand was synthesized in vitro by the use of T7 polymerase (see Fig. S1A). 4
ACCEPTED MANUSCRIPT For preparation of an antisense EGSWRN1, the forward primer 5'AAAACTGCAGAGATCTGGAAGCAGACTCTAAATC-3' and reverse primer 5'-
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CGGGGTACCTTTAAAAATGGTGCGGAAAGAAGGATTCGAACC-3' were used for amplification of
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a portion of an E. coli precursor tRNATyr gene by PCR. Italicized nucleotides appear in the EGS
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(Figure 1C). The PCR product was digested with Pst I and Kpn I, which had recognition sites designed in the primers, and the digested 70-bp EGSWRN1 DNA was cloned in pBluescript(SK)
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linearized by the same restriction enzymes (Fig. S1B). The EGSWRN1 gene construct was transcribed in vitro by T3 RNA polymerase (Fig. S1B).
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For expression of the catalytic M1 RNA in human cells, a plasmid containing a 377-bp rnpB gene was amplified by PCR using the primers 5’-GAGGAAGATCTGAAGCTGACCAG-3’ and
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5’-GACGGGGTACCAAAAAAGGTGAAACTGA-3’. The product was digested with Bgl II and Kpn I
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and cloned in pSuper under the control of a murine U6 snRNA promoter (Fig. S1C). The 316-
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bp U6 snRNA promoter was initially generated by amplification of a murine genomic DNA fragment having U6 snRNA gene by PCR using the primers 5'-
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CGGGAATTCGATCCGACGCCGCC-3' and 5'-GAAGATCTCAAACAAGGCTTTTCTC-3’ and. The PCR product was then digested with Eco RI and Bgl II and cloned in pSuper, thus replacing the original RPPH1 gene promoter (Fig. S1C). The murine U6 snRNA promoter enables strong gene transcription by human RNA polymerase III [38], which utilizes a short poly-thymidine sequence for termination, thus attaching a short 3’ uridine sequence to the resulted transcript. For expression of EGSWRN1 RNA in cells, the EGSWRN1 gene construct described above (Fig. S1B) was digested with Bgl II and Kpn I, and excised DNA insert was cloned in pSuper under the control of a murine U6 snRNA promoter (Fig. S1D). Cell cultures, transfection and staining 5
ACCEPTED MANUSCRIPT Adherent HeLa S3 and HEK293 cells were grown in a high glucose DMEM (Invitrogen) supplemented with 5% fetal bovine serum, streptomycin (100 g/ml), penicillin (100 U/ml)
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and Nystatin (12.5 U/ml). Cells were incubated in 5% CO2 at 37°C. For transfection, 1-5 × 105
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cells grown in 92 × 17 mm style petri dishes were transfected with expression or empty
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vector (15 g) in 10 ml medium using the calcium phosphate method [39]. For examination of cell viability, crystal violet staining assays were performed with cells grown in 96- or 6-well
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tissue culture plates and transfected with relevant expression vectors using the polyethylenimine (PEI) transfection reagent. Plates were scanned at O.D. 590 nm by TECAN
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Spark™ 10M multimode microplate reader. RNase protection analysis
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Total RNA (30 g) extracted from cells by the use of the Trizol reagent (Invitrogen) or acid-
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phenol RNA extraction method was subjected to RNase protection analysis [39] using 0.5 ×
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106 cpm of antisense RNA probe internally labeled with [-32P]GTP. Hybridization was performed in 40-l volume of buffer A (80% deionized formamide, 40 mM PIPES, pH 6.7, 400
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mM NaCl and 1 mM EDTA) for 14 hours at 42-46°C. RNA was then digested by adding 10 volumes of buffer B (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 300 mM NaCl) that contained 0.8 g/ml of RNase A and 60 units/ml of RNase T1. After 15 min at 22-25°C, the RNases were eliminated by Proteinase K/SDS treatment and protected RNAs were extracted with phenol:chloroform and ethanol precipitated in the presence of tRNA, as a carrier. The pellet of RNA was resuspended in a loading buffer (95% formamide, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA), heat-denatured and then separated on a 8% polyacrylamide/7M urea gel. Protected RNA bands were visualized by autoradiography. For DNA size markers, pBluescript DNA was digested with Msp I and labeled with [-32P]dCTP using the Klenow enzyme. RNase mapping analyses 6
ACCEPTED MANUSCRIPT A sense WRN mRNA was transcribed in vitro by the use of T7 RNA polymerase (Fig. S1A). The mRNA (95 pmol) was dephosphorylated by a calf intestinal phosphatase, and then was
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labeled at its 5′ termini in a phosphorylation reaction that contained 100 µCi of [-32P]ATP
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(3000 Ci/mmol) and T4 polynucleotide kinase. The labeled mRNA was separated on a 5%
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polyacrylamide/8 M urea gel and the full-length 166-nt WRN mRNA was extracted and ethanol precipitated.
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Labeled WRN mRNA (3 × 104 cpm) was subjected to partial digestion by RNase T2 (Invitrogen) or RNase T1 (Sigma) under denaturing and non-denaturing conditions. Digestion
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under denaturing condition was done in the presence of 0.2-1.0 units of RNase T1 or RNase T2 in a final volume of 20 µl in 1 × digestion buffer (20 mM Sodium Citrate, pH 5.0, 1 mM
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EDTA and 7 M Urea) for 10-30 min at 55°C. Digestion under non-denaturing conditions was
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performed in the presence of 0.001-0.005 units of RNase T1 or 0.1-0.2 units RNase T2 in 1 ×
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digestion buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 50 mM KCl, 0.2 g of tRNA) for 10 min at 37°C. The digestion reaction was stopped by the addition of a loading dye (9 M Urea,
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10% Glycerol, 0.05% Bromophenol Blue and 0.05% Xylene Cyanol ) and nucleases were eliminated by the addition of Proteinase K/SDS. RNA was extracted by phenol and precipitated by ethanol. The RNA pellet was resuspended in a loading buffer and analyzed by separation on an 8% polyacrylamide/7 M urea gel in 1 x TBE buffer. RNA ladders were prepared by hydrolysis of the WRN mRNA in an alkaline buffer (50 mM sodium bicarbonate, pH 9.0, and 1 mM EDTA trisodium salt) for 3 min in boiling water. Western blot and immunoprecipitation analyses Cell pellets were placed on ice for 5-10 min before the addition of 5 volumes of hypotonic buffer [10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 5 mM KC1, 1 mM dithiothreitol (DTT), 0.2 mM Pefabloc protease inhibitor]. After 30 min, cells were disrupted by 15-25 strokes in a 7
ACCEPTED MANUSCRIPT Dounce homogenizer and the cell debris was removed from the extract by centrifugation in an Eppendorf centrifuge at 20,000 × g at 4°C for 30 min. The soluble fraction was collected
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and stored at - 80°C. Protein concentration was determined by the Bradford method using
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Bio-Rad SmartSpec 300 photo-spectrometer.
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For Western blot analysis, proteins in whole cell extracts (S20) were separated on an 8-12% polyacrylamide/0.1% SDS gel, electro-transferred to nitrocellulose filters and
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immunoblotted with primary antibodies directed against human WRN (Bethyl Laboratories, Inc.), -Tubulin, C23, B23 (Santa Cruz Biotechnology Inc.) or actin (Sigma). A 1:28,000 dilution
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of the corresponding secondary antibodies was used. Blots were washed and bands were visualized using the ECL chemiluminescent kit, following the instructions of the manufacturer
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(Amersham). Quantitation of the values of the visualized protein bands was done by the use
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of the EZQuant-Gel software for densitometric quantitation.
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For immunoprecipitation of active WRN helicase, polyclonal anti-WRN rabbit antibodies were added to a whole cell extract (S20) at a ratio of 1:20 (v/v). The
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concentration of KCl was then adjusted to 150 mM and the 400-l mixture was subjected to nutation for overnight at 4°C. Protein A/G agarose beads (20 l), washed for three times with coupling buffer (10 mM Tris-HCl, pH 8.0, 150 mM KCl and 0.05% Igepal) were added to the mixture. After nutation for 5 h at 4°C, immunoprecipitates were prepared by centrifugation at 1,000 × g for 3 min, rinsed four times with washing buffer (40 mM Tris-HCl, pH 8.0, and 40 mM KCl) and resuspended in 20 l of 1 × exonuclease buffer (see below). Assay of WRN helicase-nuclease activity The immunoprecipitates described above were assayed for the helicase-nuclease activity of WRN on a fork DNA substrate, as described by Opresko et al. [11]. This substrate was made of the Tstem 25 deoxyoligonucleotide, 5’8
ACCEPTED MANUSCRIPT GCACTGGCCGTCGTTTTACGGTCGTGACTGGGAAAACCCTGGCG-3', annealed with a 32P-labeled Flap 26 deoxyoligonucleotide, 5'-TTTTTTTTTTTTTTTTTTTTTTCCAAGTAAAACGACGGCCAGTGC-
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3' [11]. The latter deoxyoligonucleotide (20 pmol) was labeled at its 5’ terminus by a T4
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polynucleotide kinase in the presence of 60 Ci of [-32P]ATP following the manufacturer’s
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instructions (New England BioLabs Inc.). The Flap 26 deoxyoligonucleotide was separated on an 8% polyacrylamide/7 M urea gel and extracted. The specific activity was ~ 7 × 107 cpm/g
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DNA, as determined by the TCA precipitation method. For annealing, 0.0006 pmol of Flap 26 (~ 7,000 cpm) were incubated with 0.005 pmol of Tstem 25 in 1 × TE buffer (10 mM Tris-HCl,
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pH 8.0 and 1 mM EDTA, pH 8.0) and the 20-l mixture was heated and slowly cooled. An aliquot of 3 l from the resulting fork substrate was mixed with 4-l sample of the
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immunoprecipitate described above in 1 × digestion buffer (40 mM Tris-HCl, pH 8.0, 100 M
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ZnCl2, 5 mM DTT, and 100 g/ml BSA) in a total volume of 30 l. After 1 h at 37°C, the
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digestion reaction was stopped by the addition of 50% formamide (made in 0.5 × TBE, 0.1% Bromophenol blue and 0.1% Xylene Cyanol) and products were separated on a 10%
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sequencing gel in 1 × TBE buffer and visualized by exposing the gel to autoradiography. Cleavage assay of RNase P In vitro cleavage of WRN mRNA/EGSWRN1 by M1 RNA was performed in 1 × MRP/TNET buffer that contained 80 mM MgCl2 [40]. The WRN mRNA was internally labeled with [-32P]GTP. Cleavage products were then separated on an 8% polyacrylamide/7M urea gel and visualized by autoradiography. Quantitation of the values of the RNA bands was done by the EZQuantGel software for densitometric quantitation. When a partially purified HeLa nuclear RNase P was tested in cleavage reactions , the 1 × MRP/TNET buffer contained 15 mM MgCl2 [40]. Results Delineation of single-stranded RNA regions in WRN mRNA for annealing of EGSs 9
ACCEPTED MANUSCRIPT To map single-stranded RNA regions in the human WRN mRNA that could be accessible to annealing EGS RNAs, a 5’-end labeled 166-nt WRN mRNA segment was subjected to RNase
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sensitivity mapping, as previously described (see Materials and Methods)[41]. The mRNA
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segment has 152 nt that corresponded to the WRN mRNA sequence starting from the AUG
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initiation codon, and some extra 14 nt that corresponded to the cloning plasmid (Fig. S1A). The results revealed that sequences spanning G17-G21, G32-G35 and G50-G53 of the WRN
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mRNA were sensitive to RNase T1 (Fig. 1A, lanes 7 and 8 vs 3), which attacked G residues (Fig. 1A, lanes 4-6). Likewise, sequences spanning A18-A29 and A49-A56 were accessible to
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RNase T2, which preferentially cleaved A residues (Fig. 1A, lanes 12 and 13 vs 10). Hence, several regions adjacent to the AUG codon of the WRN mRNA exist in single-stranded RNA
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conformations (Fig. 1B), which could be suitable for construction of EGSs (see below).
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Computational prediction of the secondary structure of the 5’ region of the WRN mRNA by
shown).
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the m-fold software [42] further corroborated the above nuclease mapping analyses (not
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EGS-directed cleavage of WRN mRNA by M1 RNA in vitro Based on the aforementioned mapping analyses, several custom-designed EGS RNAs directed against the A18-A29, A49-A56, G17-G21, G32-G35 and G50-G53 regions of the WRN mRNA were synthesized and tested for guiding cleavage of an internally 32P-labeled 166-nt WRN mRNA by M1 RNA in vitro (data not shown)(see Materials and Methods). A single EGS RNA, henceforth termed EGSWRN1, and which was directed against the G35-A41 region (Fig. 1C), guided M1 RNA to accurately cleave the WRN mRNA (Figs. 2A and B; also data not shown), as manifested in the generation of two major cleavage products, 5’WRN and 3’WRN, which consisted of 49 nt and 116 nt, respectively (Fig. 2A, lanes 2-6). This cleavage pattern indicated that M1 RNA excised its target WRN mRNA at the expected site between 10
ACCEPTED MANUSCRIPT A34 and G35 (Fig. 1C). Maximal cleavage was obtained at molar ratios of 150:1 and 300:1 between EGSWRN1 and WRN mRNA and in the presence of excess amounts of M1 RNA (Fig.
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2B, lanes 3-6). By contrast, the WRN mRNA was not efficiently cleaved by a purified HeLa
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nuclear RNase P [43], which was examined under appropriate reaction conditions (Figs. 2C,
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lanes 3-8, and 2D; also data not shown).
The results suggest that EGSWRN1 and WRN mRNA formed a hybrid RNA with a
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precursor tRNA-like structure susceptible to cleavage by M1 RNA at a specific position adjacent to the initiation codon of the WRN mRNA. The predicted secondary structure of the
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hybrid RNA consisted of 5’ leader, D stem, acceptor stem starting with G residue (G35) and 3’ CCA triplet (Fig. 1C). These tRNA-like features were shown to be important for recognition
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of target RNAs by M1 RNA [26,28]. The failure of HeLa RNase P to cut the WRN mRNA
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provides an advantage for selective targeting of this transcript in human cells (see below).
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EGS-directed cleavage of endogenous WRN mRNA in cells To check if EGSWRN1 can direct an accurate cleavage of endogenous WRN mRNA in human
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cells, synthetic genes coding for EGSWRN1 and M1 RNA (Fig. S1, C and D) were expressed in HeLa cells by transient transfection experiments (see Materials and Methods). RNase protection analysis revealed that the endogenous WRN mRNA, which was identified as a protected 132-nt RNA band (Fig. 3B), was excised by M1 RNA at the expected site, as manifested in the generation of two specific cleavage products, 5’WRN (35 nt) and 3’WRN (96 nt) in cells transfected with the EGSWRN1 and M1 RNA gene constructs (Fig. 3C, lane 5 vs 4; 97- and 35-nt bands). This cleavage of WRN mRNA was transient (Fig. 3C, lane 5 vs 7) and correlated with the expression of M1 RNA in cells (Fig. 3D, lanes 4 vs 6). EGSWRN1 was not detected by RNase protection and Northern blot analyses (Hitrik A., data not shown), even
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ACCEPTED MANUSCRIPT though the cleavage of the WRN mRNA was dependent on the expression of EGSWRN1 (see below). Hence, it is likely that EGSWRN1 is an unstable transcript in cells.
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In addition to the full length 132-nt WRN mRNA, smaller mRNA bands were
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protected in the control cells (Fig. 3C, lanes 4; asterisks). These bands represent degraded
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WRN mRNAs (Fig. 3C, lane 4 vs 3), whose steady state levels increased in transfected cells, in which the WRN mRNA was cleaved by M1 RNA to generate the 5’WRN and 3’WRN mRNA
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fragments (Fig. 3C, lanes 5 and 7 vs lanes 4 and 6). These two specific mRNA fragments were relatively resistant to degradation (Fig. 3C, lane 7 vs 5; 3’WRN). As the total RNAs extracted
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from cells were intact (Fig. 3A), the findings support the conclusion that WRN mRNA is an unstable transcript in HeLa cells. This conclusion is consistent with the rapid turnover
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(degradation) of the WRN protein in cancer cells [44].
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EGSWRN1-directed knockdown of the WRN protein
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Western blot analysis revealed that expression of the WRN protein was markedly reduced in HeLa cells transfected with M1 RNA and EGSWRN1 gene constructs. Thus, a reduction of > 95%
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in the steady state level of the 167-kDa WRN was seen in 24 h of transfection of cells (Figs. 4A, lane 2 vs 1, 3 and 4 and 4B). This striking reduction was transient (Fig. 4A, lane 2 vs 6; also data not shown) and concomitant with expression of M1 RNA (Fig. 3D). Moreover, WRN knockdown was efficient in cells transfected with EGSWRN1 and M1 RNA gene constructs at a molar ratio of 1:1, when compared with those obtained at ratios of 1:2 and 1:3 (Figs. 4C, lane 2 vs 3 and 4; 4D). However, neither M1 RNA nor EGSWRN1 alone facilitated efficient knockdown of WRN in control cells (Fig. 4A, lane 2 vs 3 and 4; lane 6 vs 7 and 8). The above EGSWRN1-directed WRN knockdown is not specific for a cell type. Thus, a marked reduction of > 90% in the steady state level of WRN was obtained in HEK293 cells
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ACCEPTED MANUSCRIPT transfected with EGSWRN1 and M1 RNA gene constructs for 48 and 72 h (Fig. S2A, lane 3 vs 4 and 5 vs 6).
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Inhibition of WRN helicase-nuclease activity
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Immunoprecipitation studies revealed that the 5’-3’ DNA helicase-exonuclease activity of
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WRN [9,11], which was tested on a forked DNA duplex substrate (Figs. 5B and S3)(see Materials and Methods) [11], was very low in HeLa cells with EGSWRN1-directed WRN
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knockdown, when compared with those brought down from mock-transfected (Fig. 5C, lanes 5 and 7 vs 4 and 6) or untreated (Fig. 5C, lanes 5 and 7 vs 2) cells. These enzymatic assays
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were performed in the presence of Zn+2 ions in reaction mixtures that lacked ATP (see Materials and Methods), so that to favor the exonucleolytic activity over the helicase action
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of WRN [11,12]. Likewise, a low helicase-exonuclease activity of WRN was evident in
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immunoprecipitates brought down from HEK293 cells deficient in WRN (Figs. S4, A and B).
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The results described so far show that EGSWRN1 guides M1 RNA to cleave WRN mRNA in cells, thus resulting in inhibition of translation and helicase-nuclease activity of WRN.
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WRN knockdown leads to inhibition of cell viability Crystal violet staining assays revealed that HeLa cells with WRN knockdown showed a marked decrease of ~ 60% in viability after 72 h of transfection, when compared with that of mock-transfected cells (Fig. 6A, lanes 7-9 vs lanes 10-12 and Fig. 6B; also data not shown). Cells deficient in WRN only marginally proliferated in the cultures, even after 3 days of transfection (Fig. 6, N vs L and M). By contrast, cells transfected with M1 RNA or EGSWRN1 alone showed a minor decrease of viability (Fig. 6B) and progressively proliferated from 24 to 72 h in cultures (Fig. 6, H vs F, G and K vs I, J). Based on GFP signals, the transfection efficiency was 40-60% of cultured cells (data not shown). Moreover, the crystal violet stains of cells deficient in WRN were darker and rounder than those of control cells (Fig. 6, N vs E, 13
ACCEPTED MANUSCRIPT H, K and Fig. S5). This prevalent atypical staining may reflect high percentage of nonproliferating cells that were arrested after mitosis (Fig. S5; note pairs and aggregates of
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stained nuclei). By contrast, the viability of a slowly growing HEK293 cell line deficient in
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WRN remained unaffected (data not shown).
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The results indicate that HeLa cells with EGSWRN1-directed knockdown of WRN exhibit reduced viability. This outcome may be related to severe failures of DNA repair and
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replication, thus leading to cell cycle arrest, an issue that will be addressed in future studies. Nevertheless, it has been reported by others that HeLa cells with siRNA-mediated WRN
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knockdown or WS cells undergo mitotic catastrophe [19] and apoptosis [13,18,21]. Discussion
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We have shown that the EGS/RNase P RNA system is suitable for targeting the human WRN
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helicase in cultured cell lines. Thus, our results reveal that a custom-designed EGS, termed
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EGSWRN1, can direct M1 RNA to cleave WRN mRNA at a site adjacent to the initiation codon, thus effectively inhibiting translation of WRN protein in cells. EGSWRN1-guided knockdown of
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WRN leads to inhibition of viability and proliferation of HeLa cells, but not slowly growing HEK293 cells. Accordingly, this targeting system could be useful for selective therapy of cancers, for which this DNA helicase is critical for viability and rapid proliferation. Moreover, WRN is implicated in genome maintenance and aging, and thereby, knockdown of this helicase may help in revealing new molecular mechanisms that underlie the development of cancers related to human aging. The EGS/RNase P system offers several advantages for targeting cancer genes. Thus, it exhibits high specificity, which is based on recognition of the target RNA with tRNA-like structure [26], and provides potent targeting efficacy (e. g. this study), comparable to that of siRNA [2,33]. Effective delivery of EGSs to whole model organisms is also feasible 14
ACCEPTED MANUSCRIPT [28,31,32,45,46). Moreover, in vitro selected EGSs with enhanced affinity and chemically modified EGSs with improved durability [9-11] are available for therapeutic purposes. For
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instance, EGSs can be constructed from morpholino oligonucleotides with and without
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conjugated cell-penetrating peptide and used to target bacterial and parasitic agents [47-
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49], including Staphylococcus aureus infecting cutaneous wounds in mice by using a thermoresponsive gel delivery system [37]. These advances should help in the future in
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designing modified or variants of EGSWRN1 to be assessed in therapy of tumors, possibly those confined to accessible exterior tissues of model mice and humans
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Acknowledgments
We thank Dr. Tali Burstyn-Cohen (Hebrew University, Israel) for supporting the cell biology
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section of this research. We also thank Profs. Sidney Altman (Yale University, USA), Chang-En
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Yu (University of Washington, Seattle, Washington, USA) and Ram Reddy (Baylor College of
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Medicine, Houston, Texas, USA) for providing polyclonal antibodies directed against human RNase P, WRN cDNA and U6 snRNA gene, respectively. R. Serruya and N. Orlovetskie are
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grateful for doctoral fellowship support from the Hoffman Leadership and Responsibility Program and the Rudin Foundation, respectively. This research was supported by the Israel Cancer Association (grant # 2002-0048-B) and Israel Science Foundation (grant # 368/11). References 1.
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ACCEPTED MANUSCRIPT 49. Y. Augagneur, D. Wesolowski, H.S. Tae, S. Altman, C. Ben Mamoun Gene selective mRNA cleavage inhibits the development of Plasmodium falciparum. Proc. Natl. Acad. Sci.
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ACCEPTED MANUSCRIPT Figure legends Fig. 1. RNase mapping analysis of the 5’ coding region of human WRN mRNA. (A) A 5’-end
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labeled WRN mRNA was left undigested (lanes 2 and 9) or partially digested with RNase T1
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(lanes 3-7) and RNase T2 (lanes 10-12), which were added to the reactions at the indicated
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units. The WRN mRNA existed in native (N) or denatured (D) form (see Materials and Methods). Cleavage products were separated on a 12% sequencing gel. The positions of G
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and A residues that were highly sensitive to nuclease attacks are shown. Two size markers, a ladder of a partially hydrolyzed WRN mRNA (lane 8) and pSupS1 (precursor tRNASer) partially
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digested with RNase T1 (lane 1), were included. (B) A predicted secondary structure of the first 80 nt of the WRN mRNA, starting from the AUG initiation codon (boxed). The structure
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was based on the RNase mapping analyses and computational modeling (not shown). The
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nucleotides in the WRN mRNA that were highly sensitive to attacks by RNase T1 (black
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arrowhead) and RNase T2 (white arrowhead) are specified. (C) A proposed secondary structure of the EGSWRN1/WRN mRNA hybrid. A precursor tRNA-like conformation is formed
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by annealing of the three-quarter EGSWRN1 with WRN mRNA. The UU residues that appear downstream of the CCA triplet (boxed) at the 3’ end of the EGS depict uridines added by Pol III during termination. Arrow points to the site of cleavage by M1 RNA. Numbers refer to the nucleotide positions of the WRN mRNA seen in B. Fig. 2. EGSWRN1-directed cleavage of WRN mRNA by M1 RNA in vitro. (A) Cleavage of an internally 32P-labeled WRN mRNA segment (166-nt) by M1 RNA was tested in the presence of 0, 0.15, 0.3, 0.6 and 1.2 M of EGSWRN1 (lanes 2-6) (see Materials and Methods). The expected cleavage products of 117 nt and 49 nt in length, which were resolved by electrophoresis in an 8% polyacrylamide gel, are indicated. Cleavage was not seen in the
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ACCEPTED MANUSCRIPT absence of EGSWRN1 (lane 7). An RNA size marker, generated by cleavage of a precursor tRNASer by M1 RNA, is seen in lane 1. (B) The signal intensities of the WRN mRNA and
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cleavage products bands seen in A were quantitated and plotted (see Materials and
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Methods). (C) Two highly active preparations of purified HeLa nuclear RNase P produced
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very weak cleavages of WRN mRNA annealed with EGSWRN1, which was added at concentrations of 0, 0.3, 0.6 and 1.2 M (lanes 3-5 and lanes 6-8). An RNA size marker,
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generated by cleavage of a precursor tRNASer by a DEAE-purified HeLa RNase P, is seen in
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lane 1. (D) A plot of the signal intensities of WRN mRNA and cleavage products seen in C. Fig. 3. EGSWRN1 directs M1 RNA to accurately cleave WRN mRNA in HeLa cells. (A) Total RNA
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extracted from untransfected cells (lanes 1 and 3) or cells transfected with expression
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vectors for EGSWRN1 and M1 RNA (lanes 2 and 4)(Fig. 1SC and D) was separated on an agarose gel stained with ethidium bromide. Positions of 5S, 18 and 28S rRNAs are shown. (B)
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A depiction of the mRNA fragments protected by the antisense WRN RNA probe in RNase protection analysis (See Materials and Methods). This probe protects 132 nt in the 5' coding
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region of endogenous WRN mRNA or 35-nt and 96-nt WRN mRNA fragments generated from accurate cleavage of WRN mRNA by M1 RNA. (C) RNase protection analysis of total RNAs seen in A using an internally labeled antisense WRN mRNA probe illustrated in B (see Materials and Methods). The fully protected WRN mRNA and cleavage products of 5'WRN and 3'WRN mRNAs were seen in HeLa cells transfected with M1 RNA/EGSWRN1 (ratio 1:1) for 24 and 48 h (lanes 2 and 4), but not in control cells (lanes 1 and 3). These cleavage products were separated on an 8% denaturing polyacrylamide gel. A size marker is shown in lane 8. Degradation products of the WRN mRNA are indicated by asterisks. An increase in the steady state level of endogenous WRN mRNA was seen from 24 to 48 h (lanes 1 and 3), which was
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ACCEPTED MANUSCRIPT accompanied by a similar increase in WRN protein (see Figure 5). (D) RNase protection analysis of M1 RNA expressed in HeLa cells after transfection for the indicated time points
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(lanes 2, 4 and 6). Control cells were mock-transfected with an empty vector (lanes 4, 6 and
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8). The internally 32P-labeled, antisense M1 RNA probe (lane 1) identified two specific RNAs
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of ~ 200 and 170 nt in length. These two M1 RNA fragments were reproducibly detected, possibly a result of base pair breathing or base modification of M1 RNA in cells.
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Fig. 4. EGSWRN1-directed knockdown of WRN protein by M1 RNA. (A) HeLa cells were transfected with M1 RNA gene (lanes 3 and 7), EGSWRN1 gene (lanes 4 and 8) or M1 RNA and
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EGSWRN1 genes (lanes 2 and 6). An empty vector was used as a control for transfection
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(mocked; lanes 1 and 5). After 24 and 48 h, whole HeLa extracts (S20) derived from these cells
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were prepared and equal protein amounts were analyzed for the 167-kDa WRN protein by Western blot analysis using specific antibodies against WRN (upper panel) or -tubulin (lower
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panel) (see Materials and Methods). An extract of untransfected cells was used as an additional control (lane 9). The relative signal intensities of the WRN protein bands after
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normalization with those of the -tubulin appear below the panels. (B) Graph represents the relative values of the signal intensities of WRN from three independent experiments with error bars. (C and D) HeLa cells were transfected as in A, but the ratios of the expression vectors carrying the M1 RNA and EGSWRN1 genes (see Figure S1C and D) varied as indicated. Western blot analysis and band quantitation were done as in A and B.
Fig. 5. Inhibition of the 5’-3’ helicase-exonuclease activity of WRN. (A) HeLa cells were transfected with M1 RNA and EGSWRN1 gene constructs (lanes 2 and 4) or with an empty vector (lanes 1 and 3) for 24 and 48 h. Whole extracts prepared from cells were then subjected to
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ACCEPTED MANUSCRIPT Western blot analysis using antibodies against WRN (upper panel) or -tubulin (lower panel). The relative ratios of WRN knockdown were calculated as in Figure 4A. (B) Draw of the DNA
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fork substrate, which was made of two annealing deoxyoligonucleotides, Tstem 25 and Flap
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26. Asterisk points to the 32-P labeling of the 5’ end of Flap 26. (C) Extracts described in A were
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subjected to immunoprecipitation analysis using antibodies directed against the WRN protein, and the 3'-5' DNA helicase and exonuclease activities of the immuneprecipitated WRN protein
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were examined on a labeled DNA fork substrate (lanes 4-7) (see Materials and Methods). The digestion products were separated on a sequencing gel. As controls, the fork substrate (lane 1)
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was incubated in digestion reactions containing an extract of untransfected cells (lane 2), buffer alone (lane 3), immunoprecipitate of a nonspecific antibody (lane 8),
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immunoprecipitate of a preimmune rabbit serum (lane 10) or beads alone (lane 9).
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Fig. 6. EGSWRN1-directed inhibition of cell viability. (A) Crystal violet staining of HeLa cells transfected with EGSWRN1 (1-3), M1 RNA (4-6) or EGSWRN1 and M1 RNA (7-9) gene constructs for
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24, 48 h and 72 h, as indicated. As control, cells were mock-transfected with a reporter GFP gene construct, pEGFP-C1 (10-12). Cells were cultured in a 96-well plate and transfected in triplicates using the polyethylenimine transfection reagent. (B) Graph represents the results of three independent transfection experiments performed for 72 h as in A and in which the optical densities (O.D. 580) of triplicates of stained HeLa cell cultures were measured. Error bars represent standard deviations, whereas asterisks denote the cutoffs of statistical significance of p-values. (C-N) Sample of cultured cells seen in A were visualized by microscopy. Scale bar=50 m.
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ACCEPTED MANUSCRIPT Highlights o The human WRN helicase is a potential target for cancer therapy.
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o WRN helicase is targeted in human cell lines by a custom-designed EGS/RNase P system.
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o EGS-directed knockdown of WRN is highly efficient and leads to cell death.
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o The EGS/RNase P system could be beneficial for selective cancer therapy.
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S1874-9399(16)30001-3 doi: 10.1016/j.bbagrm.2016.01.004 BBAGRM 981
To appear in:
BBA - Gene Regulatory Mechanisms
Received date: Revised date: Accepted date:
24 August 2015 29 December 2015 21 January 2016
Please cite this article as: Anna Hitrik, Ghada Abboud-Jarrous, Natalie Orlovetskie, Raphael Serruya, Nayef Jarrous, Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA, BBA - Gene Regulatory Mechanisms (2016), doi: 10.1016/j.bbagrm.2016.01.004
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
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Targeted inhibition of WRN helicase by external guide sequence and RNase P RNA
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Anna Hitrik1, Ghada Abboud-Jarrous2, Natalie Orlovetskie1, Raphael Serruya1, and Nayef
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Department of Microbiology and Molecular Genetics, The Hebrew University-Hadassah
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Medical School, Jerusalem 91120, Israel 2
Institute for Dental Sciences, The Hebrew University-Hadassah School of Dental Medicine,
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Jerusalem 91120, Israel
Corresponding author: Tel.: 972-2-6758233; Fax.: 972-2-6784010; e-mail:
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Jarrous1,2
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[email protected]
Running title: EGS-directed knockdown of WRN helicase
Keywords: External guide sequence; RNase P RNA; WRN helicase, selective cancer therapy; Werner Syndrome
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ACCEPTED MANUSCRIPT Abstract Human WRN, a RecQ helicase encoded by the Werner syndrome gene, is implicated in
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genome maintenance, including replication, recombination, excision repair and DNA damage
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response. These genetic processes and expression of WRN are concomitantly upregulated in
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many types of cancers. Therefore, targeted destruction of this helicase could be useful for elimination of cancer cells. Here, we provide a proof of concept for applying the external
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guide sequence (EGS) approach in directing an RNase P RNA to efficiently cleave the WRN mRNA in cultured human cell lines, thus abolishing translation and activity of this distinctive
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3’-5’ DNA helicase-nuclease. Remarkably, EGS-directed knockdown of WRN leads to inhibition of cell viability and proliferation. Hence, further assessment of this targeting
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system could be beneficial for selective cancer therapies, particularly in the light of the
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recent improvements introduced into EGSs.
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ACCEPTED MANUSCRIPT Introduction Werner syndrome (WS) is an autosomal recessive disease characterized by premature aging
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and increased frequency of cancer [1-4]. WS belongs to a family of progeroid syndromes,
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which include Hutchinson–Gilford progeria syndrome, Cockayne syndrome, ataxia-
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telangiectasia and Down syndrome [5,6] and is caused by mutations in the Werner Syndrome gene, WRN, which codes for a conserved RecQ helicase [1,5]. Human WRN
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protein consists of several domains that coordinate enzymatic activities, such as 3’-5’ DNA helicase and exonuclease activities [7,9], which are implicated in DNA maintenance and
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telomere preservation [1,6-10]. These enzymatic activities assist cells in removing unresolved replication intermediates and renewal of DNA replication [7]. Forked dsDNA and
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bubble DNA structures are preferred substrates for WRN [7,11], which facilitates a helicase-
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enhanced strand displacement in coordination with DNA Pol β [4,7]. WRN is also involved in
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DNA damage response via the activation of the poly(ADP-ribose) polymerase 1 [4,12-15]. These roles may explain why WS patients are predisposed to genomic instability and cancer
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[4,16]. Surprisingly, several studies showed that expression of WRN is elevated in many types of cancers, which are unrelated to those described in WS patients [4,17,18], possibly to stimulate replication progression and cell proliferation. Thus, this helicase may constitute a potential target for selective cancer therapy [4,18-20]. It has been shown that inhibition of WRN in cancer cells by small interfering RNAs (siRNAs) or small-molecule inhibitors leads to cell death via attenuation of the repair of replicative DNA defects and weakening of the DNA damage response [18-21]. Moreover, siRNA knockdown of WRN or its related RecQL1 helicase improves the chemotherapeutic efficacy of the topoisomerase inhibitor camptothecin against HeLa cells [20,23,24].
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ACCEPTED MANUSCRIPT Nonetheless, the utilization of siRNA has general limitations, such as off-target effects and instability [19,25].
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The external guide sequence (EGS)/RNase P system constitutes a powerful targeting
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approach for inhibition of gene expression [26-28]. An EGS is a short RNA designed to anneal
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with a target RNA, thus forming a hybrid RNA with a precursor tRNA-like structure that is specifically recognized and cleaved by RNase P [26-28]. RNase P is a ubiquitous
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ribonucleoprotein endoribonuclease that processes the 5' leader of precursor tRNA through recognition of structural features [26,27,29,30]. The EGS/RNase P system was shown to
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destroy various types of RNAs, pathogenic or otherwise, in cells and in model organisms [3137]. Moreover, the catalytic M1 RNA subunit of Escherichia coli RNase P, which possesses C5
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protein cofactor, functions in EGS-directed targeting of gene expression in mammalian cells
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[27,28], even though the molecular mechanism of its action is unknown.
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In this study, we report on the design and construction of an EGS RNA that guides M1 RNA to efficiently knock down WRN at the mRNA, protein and activity levels in cultured
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human cell lines. Knockdown of this DNA helicase leads to inhibition of viability of cell lines. Therefore, this targeting system constitutes a potential tool for selective cancer therapy in the future.
Materials and Methods Gene constructs A 152-bp WRN cDNA fragment, which spanned the translation initiation region, was prepared by digesting of a full length WRN cDNA [1] with Kpn I and Bam H1. The released cDNA fragment was then gel-purified and cloned in pBluescript(SK) linearized by the same restriction enzymes (Fig. S1A). The sense WRN mRNA strand was synthesized in vitro by the use of T7 polymerase (see Fig. S1A). 4
ACCEPTED MANUSCRIPT For preparation of an antisense EGSWRN1, the forward primer 5'AAAACTGCAGAGATCTGGAAGCAGACTCTAAATC-3' and reverse primer 5'-
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CGGGGTACCTTTAAAAATGGTGCGGAAAGAAGGATTCGAACC-3' were used for amplification of
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a portion of an E. coli precursor tRNATyr gene by PCR. Italicized nucleotides appear in the EGS
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(Figure 1C). The PCR product was digested with Pst I and Kpn I, which had recognition sites designed in the primers, and the digested 70-bp EGSWRN1 DNA was cloned in pBluescript(SK)
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linearized by the same restriction enzymes (Fig. S1B). The EGSWRN1 gene construct was transcribed in vitro by T3 RNA polymerase (Fig. S1B).
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For expression of the catalytic M1 RNA in human cells, a plasmid containing a 377-bp rnpB gene was amplified by PCR using the primers 5’-GAGGAAGATCTGAAGCTGACCAG-3’ and
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5’-GACGGGGTACCAAAAAAGGTGAAACTGA-3’. The product was digested with Bgl II and Kpn I
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and cloned in pSuper under the control of a murine U6 snRNA promoter (Fig. S1C). The 316-
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bp U6 snRNA promoter was initially generated by amplification of a murine genomic DNA fragment having U6 snRNA gene by PCR using the primers 5'-
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CGGGAATTCGATCCGACGCCGCC-3' and 5'-GAAGATCTCAAACAAGGCTTTTCTC-3’ and. The PCR product was then digested with Eco RI and Bgl II and cloned in pSuper, thus replacing the original RPPH1 gene promoter (Fig. S1C). The murine U6 snRNA promoter enables strong gene transcription by human RNA polymerase III [38], which utilizes a short poly-thymidine sequence for termination, thus attaching a short 3’ uridine sequence to the resulted transcript. For expression of EGSWRN1 RNA in cells, the EGSWRN1 gene construct described above (Fig. S1B) was digested with Bgl II and Kpn I, and excised DNA insert was cloned in pSuper under the control of a murine U6 snRNA promoter (Fig. S1D). Cell cultures, transfection and staining 5
ACCEPTED MANUSCRIPT Adherent HeLa S3 and HEK293 cells were grown in a high glucose DMEM (Invitrogen) supplemented with 5% fetal bovine serum, streptomycin (100 g/ml), penicillin (100 U/ml)
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and Nystatin (12.5 U/ml). Cells were incubated in 5% CO2 at 37°C. For transfection, 1-5 × 105
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cells grown in 92 × 17 mm style petri dishes were transfected with expression or empty
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vector (15 g) in 10 ml medium using the calcium phosphate method [39]. For examination of cell viability, crystal violet staining assays were performed with cells grown in 96- or 6-well
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tissue culture plates and transfected with relevant expression vectors using the polyethylenimine (PEI) transfection reagent. Plates were scanned at O.D. 590 nm by TECAN
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Spark™ 10M multimode microplate reader. RNase protection analysis
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Total RNA (30 g) extracted from cells by the use of the Trizol reagent (Invitrogen) or acid-
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phenol RNA extraction method was subjected to RNase protection analysis [39] using 0.5 ×
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106 cpm of antisense RNA probe internally labeled with [-32P]GTP. Hybridization was performed in 40-l volume of buffer A (80% deionized formamide, 40 mM PIPES, pH 6.7, 400
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mM NaCl and 1 mM EDTA) for 14 hours at 42-46°C. RNA was then digested by adding 10 volumes of buffer B (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 300 mM NaCl) that contained 0.8 g/ml of RNase A and 60 units/ml of RNase T1. After 15 min at 22-25°C, the RNases were eliminated by Proteinase K/SDS treatment and protected RNAs were extracted with phenol:chloroform and ethanol precipitated in the presence of tRNA, as a carrier. The pellet of RNA was resuspended in a loading buffer (95% formamide, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA), heat-denatured and then separated on a 8% polyacrylamide/7M urea gel. Protected RNA bands were visualized by autoradiography. For DNA size markers, pBluescript DNA was digested with Msp I and labeled with [-32P]dCTP using the Klenow enzyme. RNase mapping analyses 6
ACCEPTED MANUSCRIPT A sense WRN mRNA was transcribed in vitro by the use of T7 RNA polymerase (Fig. S1A). The mRNA (95 pmol) was dephosphorylated by a calf intestinal phosphatase, and then was
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labeled at its 5′ termini in a phosphorylation reaction that contained 100 µCi of [-32P]ATP
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(3000 Ci/mmol) and T4 polynucleotide kinase. The labeled mRNA was separated on a 5%
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polyacrylamide/8 M urea gel and the full-length 166-nt WRN mRNA was extracted and ethanol precipitated.
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Labeled WRN mRNA (3 × 104 cpm) was subjected to partial digestion by RNase T2 (Invitrogen) or RNase T1 (Sigma) under denaturing and non-denaturing conditions. Digestion
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under denaturing condition was done in the presence of 0.2-1.0 units of RNase T1 or RNase T2 in a final volume of 20 µl in 1 × digestion buffer (20 mM Sodium Citrate, pH 5.0, 1 mM
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EDTA and 7 M Urea) for 10-30 min at 55°C. Digestion under non-denaturing conditions was
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performed in the presence of 0.001-0.005 units of RNase T1 or 0.1-0.2 units RNase T2 in 1 ×
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digestion buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 50 mM KCl, 0.2 g of tRNA) for 10 min at 37°C. The digestion reaction was stopped by the addition of a loading dye (9 M Urea,
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10% Glycerol, 0.05% Bromophenol Blue and 0.05% Xylene Cyanol ) and nucleases were eliminated by the addition of Proteinase K/SDS. RNA was extracted by phenol and precipitated by ethanol. The RNA pellet was resuspended in a loading buffer and analyzed by separation on an 8% polyacrylamide/7 M urea gel in 1 x TBE buffer. RNA ladders were prepared by hydrolysis of the WRN mRNA in an alkaline buffer (50 mM sodium bicarbonate, pH 9.0, and 1 mM EDTA trisodium salt) for 3 min in boiling water. Western blot and immunoprecipitation analyses Cell pellets were placed on ice for 5-10 min before the addition of 5 volumes of hypotonic buffer [10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl2, 5 mM KC1, 1 mM dithiothreitol (DTT), 0.2 mM Pefabloc protease inhibitor]. After 30 min, cells were disrupted by 15-25 strokes in a 7
ACCEPTED MANUSCRIPT Dounce homogenizer and the cell debris was removed from the extract by centrifugation in an Eppendorf centrifuge at 20,000 × g at 4°C for 30 min. The soluble fraction was collected
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and stored at - 80°C. Protein concentration was determined by the Bradford method using
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Bio-Rad SmartSpec 300 photo-spectrometer.
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For Western blot analysis, proteins in whole cell extracts (S20) were separated on an 8-12% polyacrylamide/0.1% SDS gel, electro-transferred to nitrocellulose filters and
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immunoblotted with primary antibodies directed against human WRN (Bethyl Laboratories, Inc.), -Tubulin, C23, B23 (Santa Cruz Biotechnology Inc.) or actin (Sigma). A 1:28,000 dilution
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of the corresponding secondary antibodies was used. Blots were washed and bands were visualized using the ECL chemiluminescent kit, following the instructions of the manufacturer
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(Amersham). Quantitation of the values of the visualized protein bands was done by the use
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of the EZQuant-Gel software for densitometric quantitation.
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For immunoprecipitation of active WRN helicase, polyclonal anti-WRN rabbit antibodies were added to a whole cell extract (S20) at a ratio of 1:20 (v/v). The
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concentration of KCl was then adjusted to 150 mM and the 400-l mixture was subjected to nutation for overnight at 4°C. Protein A/G agarose beads (20 l), washed for three times with coupling buffer (10 mM Tris-HCl, pH 8.0, 150 mM KCl and 0.05% Igepal) were added to the mixture. After nutation for 5 h at 4°C, immunoprecipitates were prepared by centrifugation at 1,000 × g for 3 min, rinsed four times with washing buffer (40 mM Tris-HCl, pH 8.0, and 40 mM KCl) and resuspended in 20 l of 1 × exonuclease buffer (see below). Assay of WRN helicase-nuclease activity The immunoprecipitates described above were assayed for the helicase-nuclease activity of WRN on a fork DNA substrate, as described by Opresko et al. [11]. This substrate was made of the Tstem 25 deoxyoligonucleotide, 5’8
ACCEPTED MANUSCRIPT GCACTGGCCGTCGTTTTACGGTCGTGACTGGGAAAACCCTGGCG-3', annealed with a 32P-labeled Flap 26 deoxyoligonucleotide, 5'-TTTTTTTTTTTTTTTTTTTTTTCCAAGTAAAACGACGGCCAGTGC-
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3' [11]. The latter deoxyoligonucleotide (20 pmol) was labeled at its 5’ terminus by a T4
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polynucleotide kinase in the presence of 60 Ci of [-32P]ATP following the manufacturer’s
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instructions (New England BioLabs Inc.). The Flap 26 deoxyoligonucleotide was separated on an 8% polyacrylamide/7 M urea gel and extracted. The specific activity was ~ 7 × 107 cpm/g
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DNA, as determined by the TCA precipitation method. For annealing, 0.0006 pmol of Flap 26 (~ 7,000 cpm) were incubated with 0.005 pmol of Tstem 25 in 1 × TE buffer (10 mM Tris-HCl,
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pH 8.0 and 1 mM EDTA, pH 8.0) and the 20-l mixture was heated and slowly cooled. An aliquot of 3 l from the resulting fork substrate was mixed with 4-l sample of the
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immunoprecipitate described above in 1 × digestion buffer (40 mM Tris-HCl, pH 8.0, 100 M
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ZnCl2, 5 mM DTT, and 100 g/ml BSA) in a total volume of 30 l. After 1 h at 37°C, the
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digestion reaction was stopped by the addition of 50% formamide (made in 0.5 × TBE, 0.1% Bromophenol blue and 0.1% Xylene Cyanol) and products were separated on a 10%
AC
sequencing gel in 1 × TBE buffer and visualized by exposing the gel to autoradiography. Cleavage assay of RNase P In vitro cleavage of WRN mRNA/EGSWRN1 by M1 RNA was performed in 1 × MRP/TNET buffer that contained 80 mM MgCl2 [40]. The WRN mRNA was internally labeled with [-32P]GTP. Cleavage products were then separated on an 8% polyacrylamide/7M urea gel and visualized by autoradiography. Quantitation of the values of the RNA bands was done by the EZQuantGel software for densitometric quantitation. When a partially purified HeLa nuclear RNase P was tested in cleavage reactions , the 1 × MRP/TNET buffer contained 15 mM MgCl2 [40]. Results Delineation of single-stranded RNA regions in WRN mRNA for annealing of EGSs 9
ACCEPTED MANUSCRIPT To map single-stranded RNA regions in the human WRN mRNA that could be accessible to annealing EGS RNAs, a 5’-end labeled 166-nt WRN mRNA segment was subjected to RNase
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sensitivity mapping, as previously described (see Materials and Methods)[41]. The mRNA
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segment has 152 nt that corresponded to the WRN mRNA sequence starting from the AUG
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initiation codon, and some extra 14 nt that corresponded to the cloning plasmid (Fig. S1A). The results revealed that sequences spanning G17-G21, G32-G35 and G50-G53 of the WRN
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mRNA were sensitive to RNase T1 (Fig. 1A, lanes 7 and 8 vs 3), which attacked G residues (Fig. 1A, lanes 4-6). Likewise, sequences spanning A18-A29 and A49-A56 were accessible to
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RNase T2, which preferentially cleaved A residues (Fig. 1A, lanes 12 and 13 vs 10). Hence, several regions adjacent to the AUG codon of the WRN mRNA exist in single-stranded RNA
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conformations (Fig. 1B), which could be suitable for construction of EGSs (see below).
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Computational prediction of the secondary structure of the 5’ region of the WRN mRNA by
shown).
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the m-fold software [42] further corroborated the above nuclease mapping analyses (not
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EGS-directed cleavage of WRN mRNA by M1 RNA in vitro Based on the aforementioned mapping analyses, several custom-designed EGS RNAs directed against the A18-A29, A49-A56, G17-G21, G32-G35 and G50-G53 regions of the WRN mRNA were synthesized and tested for guiding cleavage of an internally 32P-labeled 166-nt WRN mRNA by M1 RNA in vitro (data not shown)(see Materials and Methods). A single EGS RNA, henceforth termed EGSWRN1, and which was directed against the G35-A41 region (Fig. 1C), guided M1 RNA to accurately cleave the WRN mRNA (Figs. 2A and B; also data not shown), as manifested in the generation of two major cleavage products, 5’WRN and 3’WRN, which consisted of 49 nt and 116 nt, respectively (Fig. 2A, lanes 2-6). This cleavage pattern indicated that M1 RNA excised its target WRN mRNA at the expected site between 10
ACCEPTED MANUSCRIPT A34 and G35 (Fig. 1C). Maximal cleavage was obtained at molar ratios of 150:1 and 300:1 between EGSWRN1 and WRN mRNA and in the presence of excess amounts of M1 RNA (Fig.
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2B, lanes 3-6). By contrast, the WRN mRNA was not efficiently cleaved by a purified HeLa
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nuclear RNase P [43], which was examined under appropriate reaction conditions (Figs. 2C,
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lanes 3-8, and 2D; also data not shown).
The results suggest that EGSWRN1 and WRN mRNA formed a hybrid RNA with a
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precursor tRNA-like structure susceptible to cleavage by M1 RNA at a specific position adjacent to the initiation codon of the WRN mRNA. The predicted secondary structure of the
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hybrid RNA consisted of 5’ leader, D stem, acceptor stem starting with G residue (G35) and 3’ CCA triplet (Fig. 1C). These tRNA-like features were shown to be important for recognition
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of target RNAs by M1 RNA [26,28]. The failure of HeLa RNase P to cut the WRN mRNA
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provides an advantage for selective targeting of this transcript in human cells (see below).
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EGS-directed cleavage of endogenous WRN mRNA in cells To check if EGSWRN1 can direct an accurate cleavage of endogenous WRN mRNA in human
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cells, synthetic genes coding for EGSWRN1 and M1 RNA (Fig. S1, C and D) were expressed in HeLa cells by transient transfection experiments (see Materials and Methods). RNase protection analysis revealed that the endogenous WRN mRNA, which was identified as a protected 132-nt RNA band (Fig. 3B), was excised by M1 RNA at the expected site, as manifested in the generation of two specific cleavage products, 5’WRN (35 nt) and 3’WRN (96 nt) in cells transfected with the EGSWRN1 and M1 RNA gene constructs (Fig. 3C, lane 5 vs 4; 97- and 35-nt bands). This cleavage of WRN mRNA was transient (Fig. 3C, lane 5 vs 7) and correlated with the expression of M1 RNA in cells (Fig. 3D, lanes 4 vs 6). EGSWRN1 was not detected by RNase protection and Northern blot analyses (Hitrik A., data not shown), even
11
ACCEPTED MANUSCRIPT though the cleavage of the WRN mRNA was dependent on the expression of EGSWRN1 (see below). Hence, it is likely that EGSWRN1 is an unstable transcript in cells.
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In addition to the full length 132-nt WRN mRNA, smaller mRNA bands were
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protected in the control cells (Fig. 3C, lanes 4; asterisks). These bands represent degraded
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WRN mRNAs (Fig. 3C, lane 4 vs 3), whose steady state levels increased in transfected cells, in which the WRN mRNA was cleaved by M1 RNA to generate the 5’WRN and 3’WRN mRNA
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fragments (Fig. 3C, lanes 5 and 7 vs lanes 4 and 6). These two specific mRNA fragments were relatively resistant to degradation (Fig. 3C, lane 7 vs 5; 3’WRN). As the total RNAs extracted
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from cells were intact (Fig. 3A), the findings support the conclusion that WRN mRNA is an unstable transcript in HeLa cells. This conclusion is consistent with the rapid turnover
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(degradation) of the WRN protein in cancer cells [44].
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EGSWRN1-directed knockdown of the WRN protein
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Western blot analysis revealed that expression of the WRN protein was markedly reduced in HeLa cells transfected with M1 RNA and EGSWRN1 gene constructs. Thus, a reduction of > 95%
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in the steady state level of the 167-kDa WRN was seen in 24 h of transfection of cells (Figs. 4A, lane 2 vs 1, 3 and 4 and 4B). This striking reduction was transient (Fig. 4A, lane 2 vs 6; also data not shown) and concomitant with expression of M1 RNA (Fig. 3D). Moreover, WRN knockdown was efficient in cells transfected with EGSWRN1 and M1 RNA gene constructs at a molar ratio of 1:1, when compared with those obtained at ratios of 1:2 and 1:3 (Figs. 4C, lane 2 vs 3 and 4; 4D). However, neither M1 RNA nor EGSWRN1 alone facilitated efficient knockdown of WRN in control cells (Fig. 4A, lane 2 vs 3 and 4; lane 6 vs 7 and 8). The above EGSWRN1-directed WRN knockdown is not specific for a cell type. Thus, a marked reduction of > 90% in the steady state level of WRN was obtained in HEK293 cells
12
ACCEPTED MANUSCRIPT transfected with EGSWRN1 and M1 RNA gene constructs for 48 and 72 h (Fig. S2A, lane 3 vs 4 and 5 vs 6).
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Inhibition of WRN helicase-nuclease activity
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Immunoprecipitation studies revealed that the 5’-3’ DNA helicase-exonuclease activity of
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WRN [9,11], which was tested on a forked DNA duplex substrate (Figs. 5B and S3)(see Materials and Methods) [11], was very low in HeLa cells with EGSWRN1-directed WRN
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knockdown, when compared with those brought down from mock-transfected (Fig. 5C, lanes 5 and 7 vs 4 and 6) or untreated (Fig. 5C, lanes 5 and 7 vs 2) cells. These enzymatic assays
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were performed in the presence of Zn+2 ions in reaction mixtures that lacked ATP (see Materials and Methods), so that to favor the exonucleolytic activity over the helicase action
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of WRN [11,12]. Likewise, a low helicase-exonuclease activity of WRN was evident in
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immunoprecipitates brought down from HEK293 cells deficient in WRN (Figs. S4, A and B).
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The results described so far show that EGSWRN1 guides M1 RNA to cleave WRN mRNA in cells, thus resulting in inhibition of translation and helicase-nuclease activity of WRN.
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WRN knockdown leads to inhibition of cell viability Crystal violet staining assays revealed that HeLa cells with WRN knockdown showed a marked decrease of ~ 60% in viability after 72 h of transfection, when compared with that of mock-transfected cells (Fig. 6A, lanes 7-9 vs lanes 10-12 and Fig. 6B; also data not shown). Cells deficient in WRN only marginally proliferated in the cultures, even after 3 days of transfection (Fig. 6, N vs L and M). By contrast, cells transfected with M1 RNA or EGSWRN1 alone showed a minor decrease of viability (Fig. 6B) and progressively proliferated from 24 to 72 h in cultures (Fig. 6, H vs F, G and K vs I, J). Based on GFP signals, the transfection efficiency was 40-60% of cultured cells (data not shown). Moreover, the crystal violet stains of cells deficient in WRN were darker and rounder than those of control cells (Fig. 6, N vs E, 13
ACCEPTED MANUSCRIPT H, K and Fig. S5). This prevalent atypical staining may reflect high percentage of nonproliferating cells that were arrested after mitosis (Fig. S5; note pairs and aggregates of
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stained nuclei). By contrast, the viability of a slowly growing HEK293 cell line deficient in
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WRN remained unaffected (data not shown).
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The results indicate that HeLa cells with EGSWRN1-directed knockdown of WRN exhibit reduced viability. This outcome may be related to severe failures of DNA repair and
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replication, thus leading to cell cycle arrest, an issue that will be addressed in future studies. Nevertheless, it has been reported by others that HeLa cells with siRNA-mediated WRN
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knockdown or WS cells undergo mitotic catastrophe [19] and apoptosis [13,18,21]. Discussion
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We have shown that the EGS/RNase P RNA system is suitable for targeting the human WRN
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helicase in cultured cell lines. Thus, our results reveal that a custom-designed EGS, termed
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EGSWRN1, can direct M1 RNA to cleave WRN mRNA at a site adjacent to the initiation codon, thus effectively inhibiting translation of WRN protein in cells. EGSWRN1-guided knockdown of
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WRN leads to inhibition of viability and proliferation of HeLa cells, but not slowly growing HEK293 cells. Accordingly, this targeting system could be useful for selective therapy of cancers, for which this DNA helicase is critical for viability and rapid proliferation. Moreover, WRN is implicated in genome maintenance and aging, and thereby, knockdown of this helicase may help in revealing new molecular mechanisms that underlie the development of cancers related to human aging. The EGS/RNase P system offers several advantages for targeting cancer genes. Thus, it exhibits high specificity, which is based on recognition of the target RNA with tRNA-like structure [26], and provides potent targeting efficacy (e. g. this study), comparable to that of siRNA [2,33]. Effective delivery of EGSs to whole model organisms is also feasible 14
ACCEPTED MANUSCRIPT [28,31,32,45,46). Moreover, in vitro selected EGSs with enhanced affinity and chemically modified EGSs with improved durability [9-11] are available for therapeutic purposes. For
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instance, EGSs can be constructed from morpholino oligonucleotides with and without
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conjugated cell-penetrating peptide and used to target bacterial and parasitic agents [47-
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49], including Staphylococcus aureus infecting cutaneous wounds in mice by using a thermoresponsive gel delivery system [37]. These advances should help in the future in
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designing modified or variants of EGSWRN1 to be assessed in therapy of tumors, possibly those confined to accessible exterior tissues of model mice and humans
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Acknowledgments
We thank Dr. Tali Burstyn-Cohen (Hebrew University, Israel) for supporting the cell biology
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section of this research. We also thank Profs. Sidney Altman (Yale University, USA), Chang-En
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Yu (University of Washington, Seattle, Washington, USA) and Ram Reddy (Baylor College of
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Medicine, Houston, Texas, USA) for providing polyclonal antibodies directed against human RNase P, WRN cDNA and U6 snRNA gene, respectively. R. Serruya and N. Orlovetskie are
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grateful for doctoral fellowship support from the Hoffman Leadership and Responsibility Program and the Rudin Foundation, respectively. This research was supported by the Israel Cancer Association (grant # 2002-0048-B) and Israel Science Foundation (grant # 368/11). References 1.
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ACCEPTED MANUSCRIPT Figure legends Fig. 1. RNase mapping analysis of the 5’ coding region of human WRN mRNA. (A) A 5’-end
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labeled WRN mRNA was left undigested (lanes 2 and 9) or partially digested with RNase T1
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(lanes 3-7) and RNase T2 (lanes 10-12), which were added to the reactions at the indicated
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units. The WRN mRNA existed in native (N) or denatured (D) form (see Materials and Methods). Cleavage products were separated on a 12% sequencing gel. The positions of G
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and A residues that were highly sensitive to nuclease attacks are shown. Two size markers, a ladder of a partially hydrolyzed WRN mRNA (lane 8) and pSupS1 (precursor tRNASer) partially
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digested with RNase T1 (lane 1), were included. (B) A predicted secondary structure of the first 80 nt of the WRN mRNA, starting from the AUG initiation codon (boxed). The structure
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nucleotides in the WRN mRNA that were highly sensitive to attacks by RNase T1 (black
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arrowhead) and RNase T2 (white arrowhead) are specified. (C) A proposed secondary structure of the EGSWRN1/WRN mRNA hybrid. A precursor tRNA-like conformation is formed
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by annealing of the three-quarter EGSWRN1 with WRN mRNA. The UU residues that appear downstream of the CCA triplet (boxed) at the 3’ end of the EGS depict uridines added by Pol III during termination. Arrow points to the site of cleavage by M1 RNA. Numbers refer to the nucleotide positions of the WRN mRNA seen in B. Fig. 2. EGSWRN1-directed cleavage of WRN mRNA by M1 RNA in vitro. (A) Cleavage of an internally 32P-labeled WRN mRNA segment (166-nt) by M1 RNA was tested in the presence of 0, 0.15, 0.3, 0.6 and 1.2 M of EGSWRN1 (lanes 2-6) (see Materials and Methods). The expected cleavage products of 117 nt and 49 nt in length, which were resolved by electrophoresis in an 8% polyacrylamide gel, are indicated. Cleavage was not seen in the
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ACCEPTED MANUSCRIPT absence of EGSWRN1 (lane 7). An RNA size marker, generated by cleavage of a precursor tRNASer by M1 RNA, is seen in lane 1. (B) The signal intensities of the WRN mRNA and
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Methods). (C) Two highly active preparations of purified HeLa nuclear RNase P produced
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very weak cleavages of WRN mRNA annealed with EGSWRN1, which was added at concentrations of 0, 0.3, 0.6 and 1.2 M (lanes 3-5 and lanes 6-8). An RNA size marker,
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generated by cleavage of a precursor tRNASer by a DEAE-purified HeLa RNase P, is seen in
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lane 1. (D) A plot of the signal intensities of WRN mRNA and cleavage products seen in C. Fig. 3. EGSWRN1 directs M1 RNA to accurately cleave WRN mRNA in HeLa cells. (A) Total RNA
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extracted from untransfected cells (lanes 1 and 3) or cells transfected with expression
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vectors for EGSWRN1 and M1 RNA (lanes 2 and 4)(Fig. 1SC and D) was separated on an agarose gel stained with ethidium bromide. Positions of 5S, 18 and 28S rRNAs are shown. (B)
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A depiction of the mRNA fragments protected by the antisense WRN RNA probe in RNase protection analysis (See Materials and Methods). This probe protects 132 nt in the 5' coding
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region of endogenous WRN mRNA or 35-nt and 96-nt WRN mRNA fragments generated from accurate cleavage of WRN mRNA by M1 RNA. (C) RNase protection analysis of total RNAs seen in A using an internally labeled antisense WRN mRNA probe illustrated in B (see Materials and Methods). The fully protected WRN mRNA and cleavage products of 5'WRN and 3'WRN mRNAs were seen in HeLa cells transfected with M1 RNA/EGSWRN1 (ratio 1:1) for 24 and 48 h (lanes 2 and 4), but not in control cells (lanes 1 and 3). These cleavage products were separated on an 8% denaturing polyacrylamide gel. A size marker is shown in lane 8. Degradation products of the WRN mRNA are indicated by asterisks. An increase in the steady state level of endogenous WRN mRNA was seen from 24 to 48 h (lanes 1 and 3), which was
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8). The internally 32P-labeled, antisense M1 RNA probe (lane 1) identified two specific RNAs
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of ~ 200 and 170 nt in length. These two M1 RNA fragments were reproducibly detected, possibly a result of base pair breathing or base modification of M1 RNA in cells.
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Fig. 4. EGSWRN1-directed knockdown of WRN protein by M1 RNA. (A) HeLa cells were transfected with M1 RNA gene (lanes 3 and 7), EGSWRN1 gene (lanes 4 and 8) or M1 RNA and
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EGSWRN1 genes (lanes 2 and 6). An empty vector was used as a control for transfection
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(mocked; lanes 1 and 5). After 24 and 48 h, whole HeLa extracts (S20) derived from these cells
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were prepared and equal protein amounts were analyzed for the 167-kDa WRN protein by Western blot analysis using specific antibodies against WRN (upper panel) or -tubulin (lower
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panel) (see Materials and Methods). An extract of untransfected cells was used as an additional control (lane 9). The relative signal intensities of the WRN protein bands after
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normalization with those of the -tubulin appear below the panels. (B) Graph represents the relative values of the signal intensities of WRN from three independent experiments with error bars. (C and D) HeLa cells were transfected as in A, but the ratios of the expression vectors carrying the M1 RNA and EGSWRN1 genes (see Figure S1C and D) varied as indicated. Western blot analysis and band quantitation were done as in A and B.
Fig. 5. Inhibition of the 5’-3’ helicase-exonuclease activity of WRN. (A) HeLa cells were transfected with M1 RNA and EGSWRN1 gene constructs (lanes 2 and 4) or with an empty vector (lanes 1 and 3) for 24 and 48 h. Whole extracts prepared from cells were then subjected to
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ACCEPTED MANUSCRIPT Western blot analysis using antibodies against WRN (upper panel) or -tubulin (lower panel). The relative ratios of WRN knockdown were calculated as in Figure 4A. (B) Draw of the DNA
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fork substrate, which was made of two annealing deoxyoligonucleotides, Tstem 25 and Flap
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26. Asterisk points to the 32-P labeling of the 5’ end of Flap 26. (C) Extracts described in A were
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subjected to immunoprecipitation analysis using antibodies directed against the WRN protein, and the 3'-5' DNA helicase and exonuclease activities of the immuneprecipitated WRN protein
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were examined on a labeled DNA fork substrate (lanes 4-7) (see Materials and Methods). The digestion products were separated on a sequencing gel. As controls, the fork substrate (lane 1)
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was incubated in digestion reactions containing an extract of untransfected cells (lane 2), buffer alone (lane 3), immunoprecipitate of a nonspecific antibody (lane 8),
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Fig. 6. EGSWRN1-directed inhibition of cell viability. (A) Crystal violet staining of HeLa cells transfected with EGSWRN1 (1-3), M1 RNA (4-6) or EGSWRN1 and M1 RNA (7-9) gene constructs for
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24, 48 h and 72 h, as indicated. As control, cells were mock-transfected with a reporter GFP gene construct, pEGFP-C1 (10-12). Cells were cultured in a 96-well plate and transfected in triplicates using the polyethylenimine transfection reagent. (B) Graph represents the results of three independent transfection experiments performed for 72 h as in A and in which the optical densities (O.D. 580) of triplicates of stained HeLa cell cultures were measured. Error bars represent standard deviations, whereas asterisks denote the cutoffs of statistical significance of p-values. (C-N) Sample of cultured cells seen in A were visualized by microscopy. Scale bar=50 m.
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ACCEPTED MANUSCRIPT Highlights o The human WRN helicase is a potential target for cancer therapy.
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o WRN helicase is targeted in human cell lines by a custom-designed EGS/RNase P system.
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o EGS-directed knockdown of WRN is highly efficient and leads to cell death.
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o The EGS/RNase P system could be beneficial for selective cancer therapy.
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