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To induce RNA interference (RNAi), either small interfering RNAs (siRNAs) are directly introduced into the cell or short hairpin RNAs (shRNAs) are expressed from a DNA vector. At present, shRNAs are commonly synthesized by RNA polymerase III (Pol III) promoters of the H1 and U6 RNAs. In this study, we designed and characterized a new set of plasmid vectors driven by promoters of the Epstein-Barr virus (EBV)-encoded small RNAs (EBERs). The EBERs are the most abundant transcript in infected cells and they are transcribed by Pol III. We showed that the EBER promoters were able to drive the expression of shRNA fusion transcripts. siRNAs processed from these fusion transcripts specifically and effectively inhibited the expression of homologous reporter or endogenous genes in various types of cells. The partial EBER sequences in the fusion transcripts did not activate double-stranded RNA-dependent protein kinase or suppress RNAi. In nasopharyngeal carcinoma cells, the EBER2 promoter was stronger than the H1 and U6 promoters in shRNA synthesis, leading to more effective knockdown of the target genes. Taken together, our findings suggest that the EBER promoters fundamentally different from those of H1 and U6 can be used to drive the intracellular expression of shRNAs for effective silencing of target genes in mammalian cells and particularly, in EBV-infected cells.
Gene Therapy (2008) 15, 191202 & 2008 Nature Publishing Group All rights reserved 0969-7128/08 $30.00 http://www.nature.com/gt
Web End =www.nature.com/gt
EY-W Choy1, K-H Kok1, SW Tsao2 and D-Y Jin1
1Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, Hong Kong, China and 2Department of Anatomy, Faculty of Medicine, The University of Hong Kong, Hong Kong, China
homologous reporter or endogenous genes in various types of cells. The partial EBER sequences in the fusion transcripts did not activate double-stranded RNA-dependent protein kinase or suppress RNAi. In nasopharyngeal carcinoma cells, the EBER2 promoter was stronger than the H1 and U6 promoters in shRNA synthesis, leading to more effective knockdown of the target genes. Taken together, our ndings suggest that the EBER promoters fundamentally different from those of H1 and U6 can be used to drive the intracellular expression of shRNAs for effective silencing of target genes in mammalian cells and particularly, in EBV-infected cells. Gene Therapy (2008) 15, 191202; doi:http://dx.doi.org/10.1038/sj.gt.3303055
Web End =10.1038/sj.gt.3303055 ; published online 1 November 2007
Keywords: RNA interference; EpsteinBarr virus (EBV); EpsteinBarr virus-encoded small RNA (EBER); small interfering RNA (siRNA); RNA polymerase III-dependent promoter
elicit RNAi 100-fold more potently than the corresponding 21-nucleotide siRNAs.17
Although some RNA polymerase II (Pol II) promoters have been used to express shRNAs in mammalian cells,18,19 at present shRNAs are more commonly
transcribed by mammalian U6 or H1 promoters.916 The
U6 and H1 promoters belong to type III RNA polymerase
III (Pol III) promoters and have promoter elements located extragenically.20 In addition, type II Pol III promoters, such as the tRNA promoters having promoter elements located intragenically, can also be used to drive shRNA expression.21,22 Since the intragenic promoter
elements of these promoters are co-transcribed as the 50 end of the shRNA, the secondary structure formed may confer extra stability to the overall shRNA and increase its accessibility to Dicer in the initiation step of RNAi.
Animal viruses such as EpsteinBarr virus (EBV) encode various forms of small RNAs including micro-RNAs.2325 While the biological functions of most viral small RNAs remain elusive, some of these RNAs, such as adenovirus VAI and EBV-encoded small RNAs (EBERs), are exceedingly abundant in infected cells.2628 In light of
this, we sought to investigate whether the promoters of viral small RNAs such as EBERs can be used to drive the expression of shRNAs in cultured human cells.
The EBERs with a copy number of approximately 107 per cell are by far the most abundant RNAs in EBV-infected cells.27 EBER1 and EBER2 have 165 and 169 Received 2 May 2007; revised 13 September 2007; accepted 13September 2007; published online 1 November 2007
ORIGINAL ARTICLE
Utility of EpsteinBarr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs
To induce RNA interference (RNAi), either small interfering
RNAs (siRNAs) are directly introduced into the cell or short hairpin RNAs (shRNAs) are expressed from a DNA vector. At present, shRNAs are commonly synthesized by RNA polymerase III (Pol III) promoters of the H1 and U6 RNAs. In this study, we designed and characterized a new set of plasmid vectors driven by promoters of the EpsteinBarr virus (EBV)-encoded small RNAs (EBERs). The EBERs are the most abundant transcript in infected cells and they are transcribed by Pol III. We showed that the EBER promoters were able to drive the expression of shRNA fusion transcripts. siRNAs processed from these fusion transcripts specically and effectively inhibited the expression of
Introduction
RNA interference (RNAi) is an evolutionary conserved gene-silencing mechanism, in which sequence-specic degradation of the homologous mRNA is triggered by double-stranded RNA (dsRNA).13 This phenomenon was rst discovered in Caenorhabditis elegans by injecting long dsRNA.1 However, introduction of dsRNA longer than 30 bp into mammalian cells induces the interferon (IFN) response, in which the activation of dsRNA-dependent protein kinase (PKR) and 20,50-oligoadenylate synthetase (20,50-AS) results in nonspecic RNA degradation.46 To circumvent this pathway, specic gene silencing can be achieved by direct introduction of either chemically synthesized or in vitro transcribed small interfering RNAs (siRNAs) of 21 nucleotides in length.79 Alternatively, short hairpin RNAs (shRNAs) can be expressed from a DNA vector and subsequently processed into functional siRNAs in the cell by Dicer ribonuclease.916 It is noteworthy that 27-nucleotide long synthetic dsRNAs, which are substrates of Dicer, can
Correspondence: Dr D-Y Jin, Department of Biochemistry, The University of Hong Kong, 3rd Floor, Laboratory Block, Faculty of Medicine Building, 21 Sassoon Road, Pokfulam, Hong Kong, China. E-mail: mailto:[email protected]
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nucleotides, respectively. The EBER promoters are transcribed by Pol III but they are also regulated by transcription factors Sp1 and activating transcription factor (ATF) that bind normally to Pol II promoters.27
They contain both extragenic and intragenic promoter elements. The extragenic elements include Sp1 element, ATF motif and EBER TATA box (ETAB), whereas boxes A and B are intragenic.2729 The activity of the EBER promoters is dictated by these unique features and their exceedingly high activity in EBV-infected cells is attributed to phosphorylation of ATF2 and induction of Pol IIIspecic basal transcription factors TFIIIC and BDP1.30
In this study, we designed, constructed and characterized a new set of plasmid vectors driven by the EBER promoters. We showed that these promoters were able to drive the synthesis of shRNA fusion transcripts efciently. In addition, the siRNAs processed from the shRNA fusion transcripts inhibited the expression of reporter and endogenous genes in various types of transiently or stably transfected cells. Importantly, the gene-silencing effect was specic and PKR activation was not induced. Notably, the EBER2-shRNAs induced the most pronounced gene knockdown effect in nasopharyngeal carcinoma cells. Thus, we provided the proof-of-principle that the EBER promoters can be used to drive intracellular expression of shRNAs for the
induction of RNAi. These vectors based on the EBER promoters are a useful tool for the delivery of gene-silencing agents into mammalian cells and for developing nucleic acid therapeutics.
Results
Construction of shRNA expression vectors driven by EBER promoters The EBER promoters are strong viral promoters transcribed by Pol III.27 Similar to tRNA promoters, these viral promoters contain intragenic elements that are transcribed into RNA. The highly structured transcripts produced could be more resistant to nonspecic degradation.22 In addition, the Sp1- and ATF-binding elements in the EBER promoters may further enhance transcription.28,30 In light of all these unique features of the EBER
promoters, we asked whether they could be utilized to drive the expression of shRNAs in mammalian cells. As a rst step, we set out to construct shRNA expression vectors based on the EBER promoters.
Using PCR cloning, we were able to assemble two shRNA expression vectors pEBER1-shRNA and pEBER2-shRNA controlled by EBER1 and EBER2 promoters, respectively (Figure 1a). These vectors have incorporated
Figure 1 Short hairpin RNA (shRNA) expression vectors based on EpsteinBarr virus (EBV)-encoded small RNA (EBER) promoters. (a) Schematic diagram of shRNA expression vectors. All vectors can drive the expression of shRNA (sense-loop-antisense: ; 68 nucleotides), which is terminated by a stretch of six thymidines. Three restriction sites (XbaI, ClaI and XhoI) were inserted upstream of the shRNA sequence to facilitate subcloning. pEBER1-shRNA and pEBER2-shRNA have incorporated all the extragenic (Sp1, activating transcription factor (ATF) and EBER TATA box (ETAB)) and intragenic (boxes A and B) promoter elements of the EBER promoters (B120 nucleotides). DSE: distal sequence element. PSE: proximal sequence element. (b) Schematic diagram of an RNA transcript (shRluc) expressed from pEBER2-shRNA. G:U wobble base pairing was introduced into the stem of shRluc to facilitate PCR cloning. Altered nucleotides in EBER2-shRluc m1 are circled, and altered residues in EBER2-shRluc m2 are squared.
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all the EBER extragenic and intragenic promoter elements including Sp1 element, ATF motif, ETAB, boxes A and B. shRNAs were placed downstream of the promoter through multiple cloning sites. The transcription of 29-mer shRNA is terminated with a stretch of six thymi-dines. The shRNA sequence has an eight-nucleotide loop sequence with a HindIII restriction site situated between the sense and antisense strands of the siRNA sequence. As such, the fusion transcripts produced from these vectors are EBER1-shRNA of 194 nucleotides and EBER2-shRNA of 209 nucleotides, which contain a 68-nucleotide shRNA sequence (Figure 1b) at the 30 end of the B120-nucleotide EBER1/2 RNAs. For comparison, we also constructed a series of shRNA expression vectors under the control of human tRNAVal, U6 and H1
promoters (Figure 1a).
Production of siRNAs derived from EBER-shRNA fusion transcriptsNext, we investigated whether the expression vectors driven by the EBER promoters might be used to produce shRNAs in cultured human cells. For this purpose, we chose an shRNA directed against rey luciferase (shFluc), whose effectiveness in knocking down the expression of its target gene has been documented.9,31,32
The shFluc sequence was subcloned into pEBER1-shRNA and pEBER2-shRNA vectors. To directly analyze the EBER-shRNA fusion transcripts expressed from these vectors, northern blotting was performed (Figure 2a). As expected, ample amounts of EBER-shRluc RNAs of the correct size (194 nucleotides for EBER1-shRluc and 209 nucleotides for EBER2-shRluc) were detected in transfected cells (Figure 2a, lanes 2 and 3 as compared to lane1).
shRNAs expressed in cells are thought to be processed by Dicer ribonuclease to generate siRNAs, which induce RNAi effect. Above we demonstrated the expression of EBER-shRNAs (Figure 2a). However, it remained to be determined whether siRNAs could be produced from
these EBER-shRNA transcripts. To address this issue, we performed RNase protection assay using enriched small RNAs prepared from HEK293 cells transfected with shRluc expression vectors driven by the EBER and U6 promoters (Figure 2b). We noted that the steady-state amounts of shRNA in cells transfected individually with pEBER1-shRluc, pEBER2-shRluc or pU6-shRluc were comparable (Figure 2b, upper panel, lanes 2 and 3 as compared to lane 4). Importantly, siRNAs specically hybridized to the Renilla luciferase (Rluc) sense probe were found in cells transfected with pEBER1-shRluc or pEBER2-shRluc (Figure 2b, upper panel, lanes 2 and 3). siRNAs were also detected in cells carrying pU6-shRluc (Figure 2b, upper panel, lane 4), but not in cells receiving pLuc reporter plasmids alone (lane 1). Thus, effector siRNAs were generated from the EBER-shRluc fusion transcripts.
Induction of RNAi by EBER-shRNA transcripts Detection of siRNAs in pEBER-shRNA-transfected cells (Figure 2b) predicts that specic RNAi effect could be induced. To provide evidence for this hypothesis, we cotransfected pEBER-shRNA vectors into HeLa cells together with rey luciferase (Fluc) and Renilla lucifer-ase (Rluc) reporter plasmids. The transfected cells were assayed for Fluc and Rluc activities. In this dual luciferase assay, we used pSHAG-Ff1 plasmid, which contains U6 promoter and has been shown to express shFluc efciently in cultured cells,9,31,32 as a positive
control.
As shown in Figure 3a, Fluc activity was signicantly reduced in cells transfected with pEBER1-shFluc or pEBER2-shFluc. A 70% inhibition of Fluc activity was achieved in pEBER2-shFluc-transfected cells. A similar reduction was also seen in cells carrying pSHAG-F. In contrast, cells having empty vector pEBER1-T6 or pEBER2-T6 were able to express Fluc to the same level as cells transfected with reporter plasmids alone (pFluc). Notably, the readouts of Rluc activity in cells carrying
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Figure 2 Analysis of EpsteinBarr virus (EBV)-encoded small RNA (EBER)-short hairpin RNAs (shRNAs) and small interfering RNAs (siRNAs) in cells transfected with pEBER1-shRNA targeting Rluc (shRluc) and pEBER2-shRluc vectors. (a) Northern blotting. Total cellular RNA was extracted from HeLa cells transfected with the indicated expression plasmids. (b) RNase protection assay. RNA was extracted and enriched from HEK293 cells transfected with the indicated expression plasmids. Solution hybridization and RNase treatment were carried out, followed by analysis of protected fragments on a 15% urea gel. Because a 32P-labeled probe of 29 nucleotides was used, protected fragments of siRNA contained 21 or 22 nucleotides, while all protected fragments of shRNA contained 29 nucleotides (see diagrams on the right for reference). To ensure equal loading, 5S rRNA was also stained. nt: nucleotides.
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Figure 3 Gene-silencing activity of shRNAs expressed from pEBER1-shRNA and pEBER2-shRNA vectors. (a) Silencing of rey luciferase (Fluc) expression. HeLa cells were transfected with luciferase reporter plasmids pFluc alone or pFluc plus the indicated expression vectors. The relative Fluc activity was obtained by normalizing Fluc readouts with those of Renilla luciferase (Rluc). The relative Fluc activity recovered from cells receiving pFluc alone was set as 100%. Results represent the average of triplicate experiments and the error bars indicate s.d. (b) Inhibition of Rluc expression. pEBER1-shRluc and pEBER2-shRluc were expression vectors for shRNA targeting Rluc (shRluc) driven by EpsteinBarr virus (EBV)-encoded small RNA (EBER) promoters. The relative Rluc activity was obtained by normalizing Rluc readouts with those of Fluc. The relative Rluc activity recovered from cells transfected with pRluc alone was set as 100%. Results represent the average of triplicate experiments and the error bars indicate s.d. (c) Gene-specic RNAi activity of EBER-shRluc. Cells were transfected with the Fluc and lacZ reporter plasmids alone (reporters alone) or the reporter plasmids plus the indicated expression vectors. The relative b-galactosidase activity was obtained by normalizing b-galactosidase activity readouts with those of Fluc. The relative b-galactosidase activity of cells having reporters alone was set as 100%. Results represent the average of triplicate experiments and the error bars indicate s.d. (d) Sequence-specic RNAi activity of EBER2-shRluc. An expression vector for scrambled shRNA, pEBER2-shSC, was used as a negative control and set as 100%. The shRluc stem region in EBER2-shRluc m1 and m2 mutants contains point mutations as indicated in Figure 1b.
pEBER1-shFluc, pEBER2-shFluc or pSHAG-Ff1 did not drop signicantly (data not shown), indicating that the reduction in Fluc activity was rather specic. These results suggest that the EBER promoters can be used to express shRNAs.
To further verify this, we employed the EBER promoters to express another shRNA targeting Rluc (shRluc) but not Fluc. In this case, signicant knockdown of Rluc activity was observed in HeLa cells transfected with pEBER1-shRluc or pEBER2-shRluc, when compared to cells carrying plasmid pU6-shRluc, a positive control for expression of shRluc.31,32 The inhibition of Rluc activity attributed to plasmid pEBER2-shRluc was 460% (Figure 3b). This inhibition was not seen in cells carrying reporter plasmids alone (pRluc) or an empty vector (pEBER1-T6 or pEBER2-T6). In addition, the original readouts of Fluc activity in cells receiving pEBER1-shRluc, pEBER2-shRluc or pU6-shRluc were comparable to those in cells carrying pFluc/pRluc reporter plasmids
alone (data not shown). Consistent with this, the b-galactosidase activity in cells transfected with pEBER1-shRluc and pEBER2-shRluc did not decrease when compared with cells bearing Fluc and lacZ reporters alone (Figure 3c), further demonstrating the specicity of RNAi effect. Finally, when point mutations were introduced into the stem of the shRluc (Figure 1b), the gene-silencing effect was abolished (Figure 3d). Thus, EBERshRluc RNAs were able to mediate gene- and sequence-specic silencing in mammalian cells.
The fusion transcripts produced from pEBER1-shRluc and pEBER2-shRluc contain partial EBER1 and EBER2 sequences at the 50 end (Figure 1a). While the function of EBER RNAs remains obscure, they have been shown to interact with cellular partners including PKR, L22 and La.3335 EBER RNAs can also inhibit apoptosis induced
by various signals including IFN-a, but it remains controversial as to whether this antiapoptotic activity is mediated through the inhibition of PKR.3638 To address
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Figure 4 Inuence of the partial EpsteinBarr virus (EBV)-encoded small RNA (EBER) sequences on dsRNA-dependent protein kinase (PKR) activation and RNAi. (a) Phosphorylation of PKR. Increasing amounts of the indicated plasmids were transfected into HeLa cells. For the positive control group, 100 mgml 1 of poly (I:C) was added directly to the medium. The negative control group received only the buffer that was used to dissolve poly (I:C). (b) Activation of eIF2a. pATF4-UTR-Luc is a rey luciferase (Fluc) reporter with the 50 UTR of human ATF4 fused to the coding region of Fluc. When eIF2a is activated, stimulation of ATF4 translation through upstream open reading frames in the 50 UTR can be observed, as shown in the positive control group, in which 600 nM thapsigargin (Tg) was added to the medium. The negative control group received dimethyl sulfoxide (DMSO) only. The relative Fluc activity was obtained by normalizing with the readings of the Renilla luciferase (Rluc) activity. Results represent the average of triplicate experiments and the error bars indicate s.d. (c) Inuence of EBER RNAs on RNAi. Indicated amounts of the different expression vectors were transfected into 293 T cells. Results represent the average of triplicate experiments and the error bars indicate s.d.
whether the partial EBER sequences could activate PKR, we carried out two experiments. First, we checked for the phosphorylated and active form of PKR in pEBER1/2-shRNA-transfected cells (Figure 4a). While treatment with poly(I:C) potently induced phosphorylation of PKR (Figure 4a, lane 2 as compared to lane 1), the steady-state levels of phosphorylated PKR were unchanged in pEBER-shRluc-expressing cells (Figure 4a, lanes 58 as compared to lane 1). In this setting, the shRluc expressed from U6 promoter had a weak stimulatory effect on PKR (Figure 4a, lane 4 as compared to lanes 3 and 1). In the second experiment, we assessed the activation of eukaryotic initiation factor 2a (eIF2a) using the pATF4-UTR-Luc reporter construct. eIF2a is a substrate of PKR and phosphorylation of eIF2a stimulates the translation of ATF4 through two upstream open reading frames in the 50 untranslated region (UTR).5,3941 Based on these previous ndings, the pATF4-UTR-Luc construct was used as an indicator of eIF2a phosphorylation and activation in our experiment.41 We noted that treatment with thapsigargin, a known stimulator of eIF2a kinase,5
induced a two-fold activation of luciferase reporter activity controlled by the 50 UTR of ATF4 (Figure 4b). In contrast, no activation of this reporter and thus the activity of eIF2a were observed in cells expressing EBER1-shRluc or EBER2-shRluc (Figure 4b). Hence, our two experiments consistently demonstrated that the partial EBER sequences did not activate PKR in mammalian cells.
On the other hand, EBER RNAs are highly structured42 and resemble adenovirus VAI RNA and human immunodeciency virus TAR RNA, which have recently been found to be inhibitors of RNAi by exhausting Dicer and sequestering dsRNA-binding protein TRBP, respectively.43,44 In light of this, we carried out additional
experiments to address whether EBER RNAs might inuence RNAi. Results from our control experiments shown in Figures 3a and b indicated that the partial EBER sequences produced from pEBER1-T6 and pEBER2-T6 had minimal inuence on the expression of luciferase reporter. To determine the effect of EBER RNAs on the activity of shRluc, we co-expressed
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different combinations of RNAs in 293 T cells. In our experimental setting, adenovirus VAI was fully competent in suppressing the activity of shRluc (Figure 4c; groups 3 and 4 as compared to groups 1 and 2). In contrast, neither full-length (expressed from pEBER) nor partial (expressed from pEBER1/2-T6) EBER1/2 sequences inhibited the suppressive activity of shRluc (Figure 4c; groups 3 and 4 as compared to groups 1 and2). These results suggested that EBER RNAs unlikely acted as viral suppressors of RNAi. Thus, the suppressive activities of fusion transcripts EBER1-shRluc and EBER2-shRluc on reporter expression were attributed to the shRluc sequences at the 30 end, but not to the partial EBER sequences.
Comparison of EBER2 promoter with other Pol III promoters for shRNA expressionAbove, we showed that EBER promoters were able to drive the expression of shRNAs in HeLa cells (Figure 3). To assess the utility of this type of viral promoters in different contexts, we extended our analysis to other cell lines and made a comparison between EBER2 promoter and other human Pol III promoters commonly used in shRNA expression. All these promoters (that is, EBER2, tRNAVal, U6 and H1 promoters) were inserted into the same vector backbone (Figure 1a). These shRNA expression vectors were then compared for their shRNA-mediated gene-silencing activities in four different cell lines. The pEBER2-shSC vector expressing scrambled shRNAs was used as a negative control.
The four cell lines used were HeLa (human epithelial cervical carcinoma), HEK293 (human embryonic kidney epithelial cells), C666-1 (human nasopharyngeal carcinoma) and HK1 (human nasopharyngeal carcinoma). The C666-1 cell line was derived from undifferentiated nasopharyngeal carcinoma and it consistently harbors EBV, resembling the EBV latency II status.45 The HK1 cell line was derived from well-differentiated nasopharyngeal carcinoma and it does not carry EBV.46 The
expression of EBER2 genes in the EBV+ C666-1 cells as well as the EBV HK1 cells was examined by RTPCR (Figure 5, inset). Judging from the gene-silencing activities in all four cell lines, the expression of shRluc from EBER2 promoter was more effective than from the other commonly used Pol III promoters including tRNAVal, U6 and H1 promoters (Figure 5). As such, pEBER2-shRluc was able to sustain a 6080% knockdown of Rluc activity and among all vectors tested it delivered the greatest gene-silencing effect in nasopharyngeal carcinoma cell lines C666-1 and HK1.
Analysis of mRNA target in cells carrying EBER vectors Results from dual luciferase assays support the notion that the EBER promoters can be used to drive the expression of shRNAs at least as effectively as the commonly used H1, U6 and tRNAVal promoters and in many cases worked even better (Figures 3 and 5). While reporter assays are sensitive, they only indirectly reect the gene-silencing activities of siRNAs derived from the fusion EBER-shRNA transcripts (Figure 2). The detection of strong RNAi effect induced by ERER-shRNAs and the detection of siRNAs in cells expressing EBER-shRNAs prompted us to ask whether the gene-silencing effect observed was due to siRNA-induced degradation of the target mRNA. To address this question, we performed quantitative RTPCR using total RNA extracted from pEBER1-shRluc- and pEBER2-shRluc-transfected HEK293 cells (Figure 6). Another siRNA expression plasmid pU6-shRluc was chosen for comparison. Additionally, an empty siRNA expression vector pGEM-U6 was used as a negative control. We noted that the relative Rluc mRNA level did decrease in pEBER1-shRluctransfected cells. The Rluc mRNA level dropped to a more signicant degree in cells having pEBER2-shRluc. Around 85% of the targeted Rluc mRNA was cleaved and this ratio was comparable to that observed in pU6-shRluc-transfected cells. Hence, shRNAs transcribed from vectors with the EBER promoters were effectively processed into siRNAs that induce sequence-specic mRNA degradation.
Figure 5 Comparison of shRNA expression vectors. Four different cell lines (HeLa, HEK293, C666-1 and HK1) were transfected with reporter plasmids pRluc and the indicated shRNA expression vectors (see Figure 1 for reference). Results were normalized to Fluc activity and the average of triplicate experiments was shown. The error bars indicate s.d. The relative Renilla luciferase (Rluc) activity recovered from cells with pEBER2-shSC, a vector having scrambled shRNA sequence expressed, was set as 100%. Shown in the inset are results from RTPCR analysis of EBER2 and glyceraldehydes-3-phosphate dehydrogenase (GAPDH) genes performed with EBV HK1 cells, EBV+ C666-1 cells, as well as C666-1 cells without the addition of reverse transcriptase (RT) (C666-1 no RT).
Figure 6 Quantitative PCR analysis of messenger ribonucleic acid (mRNA) degradation induced by shRNAs expressed from pEBER1-shRNA targeting Rluc (shRluc) and pEBER2-shRluc vectors. Each bar represents the average of triplicate experiments and s.d. was also plotted. Normalization with the Fluc mRNA level was carried out to obtain the relative Rluc mRNA level. The relative Rluc mRNA level of pLuc-transfected cells was set as 100%.
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Figure 7 Knockdown of cell-endogenous Nup155 transcript using shRNA-expressing vector driven by the EpsteinBarr virus-encoded small RNA 2 (EBER2) promoter. (a) Western blotting using 293 T cells transiently transfected with the shRNA-expressing vector. The relative Nup155 protein amount for each sample was analyzed using Scion Image software. (b) Western blotting using HeLa cells stably harboring the shRNA-expressing vector.
Silencing of an endogenous transcript with EBER2 vector After checking for the silencing of exogenously introduced reporter genes with a series of assays, we were also interested in assessing the effectiveness of the EBER vectors in delivering siRNAs that target cell-endogenous transcripts. We designed two independent shRNAs against human nucleoporin Nup155, a component of the nuclear pore complex required for the formation of nuclear membrane.47 In 293 T cells transfected with pEBER2-shNup155 vector, the expression of Nup155 protein was diminished by approximately 50% (Figure 7a, lanes 2 and 3 as compared to lane 1). Remarkably, when the two shNup155s expressed from the EBER2 promoter were compared to each other, their efciencies in gene silencing were found to be similar (Figure 7a, lanes 2 as compared to lane 3). In addition, when shNup155 no. 2 was separately expressed from the EBER2 and the U6 promoters, the silencing efciencies were also comparable (Figure 7a, lane 3 as compared to lane 5). Thus, the EBER2 promoter is highly efcient in driving the expression of shNup155s.
Because the above assays on the EBER2 promoter were carried out via transient transfection of cells, it will be of interest to see whether the expression of shRNAs from the EBER2 promoter could be sustained in established cell lines stably carrying the shRNA expression vector. Thus, we transfected the EBER2 promoter-based expression vector into HeLa cells and selected stable clones with the addition of G418. Western blot analyses indicated that the expression of Nup155 was constitu-
tively suppressed in several clones. It is noteworthy that this inhibition of Nup155 expression is not restricted to a single clone. For example, the amount of Nup155 recovered from two representative stable clones carrying pEBER2-shNup155 (clones nos. 2 and 3) was signicantly less than from cells containing pEBER2-shRluc (Figure 7b, lane 2 as compared to lane 1, and lane 4 as compared to lane 3). Hence, the EBER2 vector was able to deliver siRNAs targeting exogenous or endogenous transcripts into cultured mammalian cells both efciently and sustainably.
Discussion
In this feasibility study, we established the utility of the EBER promoters from EBV for the expression of gene-silencing RNAs in cultured mammalian cells. EBERshRNAs of the correct size were abundantly transcribed from the EBER promoters (Figure 2a) and were subsequently processed presumably by endogenous Dicer nuclease into siRNAs (Figure 2b). The effector siRNAs were able to induce sequence-specic degradation of target mRNA (Figure 6) leading ultimately to the knockdown of protein expression as indicated in the reduction of protein amount and activity (Figures 3 and7). The gene-silencing effect observed was gene- and sequence specic, with no evidence of PKR activation (Figures 3 and 4). The expression of shRNAs by the EBER promoters was as effective as or even more effective than that from other commonly used Pol III promoters. In addition, the EBER2 promoter was the most efcient among all promoters tested in nasopharyngeal carcinoma cells (Figure 5).
Ever since the discovery that 21-nucelotide siRNAs introduced into mammalian cells effectively induce RNAi without provoking the IFN response,7 various strategies have been used to achieve the greatest and the most stable gene-silencing effect within the cells. Although some Pol II promoters including CMV and U1 promoters18,19 have been shown to be able to deliver
shRNAs into the cells, up till now one common design of RNAi experiments is still to express shRNAs from a DNA vector driven by Pol III promoters, such as those of U6, H1 and tRNA.9,1116,21,22 Our work has added one
important group of viral Pol III promoters to the list of promoters that can drive shRNA expression. Our ndings establish the concept that shRNA fusion transcripts produced by Pol III-dependent viral promoters are effectively processed by Dicer into functional siRNAs.
While the existing pEBER-shRNA vectors are already powerful, they can be further modied and improved in several aspects. First, because efcient transcription from the EBER promoters depends on the secondary structure of the RNA,48 further modication of the EBER-shRNA sequences might enhance their transcription from the EBER promoters. Second, other unique features of the EBER promoters can also be utilized to enhance RNA expression from the pEBER-shRNA vectors. Particularly, the EBER promoters have both extragenic (Sp1, ATF and ETAB) and intragenic promoter elements (boxes A andB).2729 All these elements are important for the transcriptional activity2729 and were incorporated into
the existing pEBER-shRNA vectors (Figure 1a). The
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extragenic promoter elements of EBER may provide an opportunity for developing inducible shRNA expression vector. Third, since the activation of ATF2 and the elevation of pol III-specic transcription factors in EBV-infected cells are responsible for the exceedingly strong activity of the EBER promoters,30 RNA expression could be optimized through manipulation of the host cell. For instance, constitutively active versions of ATF2 and other key factors could be stably introduced to the cell in order to maximize RNA production. Finally, it will be of interest to see whether the EBER promoters might still be feasible for the expression of small RNAs when incorporated into viral vectors, such as lentiviral, adenoviral and even EBV-based vectors.
The EBERs localize to the nucleus.35,49 It will thus be of
interest to determine the subcellular localization of EBER-shRNAs that are transcribed from the EBER promoters. In this regard, some shRNAs expressed from the U6 promoter have been found mainly in the nucleus.22,50 The subcellular localization of RNAs expressed from Pol III promoters depends mainly on the expression cassette.50 Interestingly, while RNAi mediated by mRNA degradation occurs in the cytoplasm, siRNAs can also localize to the nucleus and induce specic RNA degradation there.51,52 In addition, nuclear siRNAs are capable of inducing specic gene silencing.5255 Thus,
shRNAs and siRNAs targeted to the cytoplasm and the nucleus can be used to knockdown different RNAs specically through different mechanisms.
EBV is etiologically associated with several lymphoid and epithelial malignancies including Burkitts lymphoma, Hodgkins disease, nasopharyngeal carcinoma and gastric cancer.56,57 While EBV can produce lytic and
persistent infection, all EBV-associated cancers involve the establishment of three types (IIII) of latency in target cells. The EBERs are most abundantly found in all types of EBV-infected cells, having 107 copies per cell.27 Previous attempts to design anti-EBV agents based on the specic and high expression of EBERs have already been successful. For example, an engineered recombinant adenovirus which depends on EBERs for replication showed specic oncolytic activity in EBV+ tumor cells.58 In our experiments, EBER-shRNAs transcribed from the EBER promoters were highly abundant in cells (Figure 2a). The degradation of target mRNA induced by these shRNAs was also remarkable (Figure 6). In addition, the EBER2 promoter was indeed most potent in nasopharyngeal carcinoma cells C666-1 and HK1 (Figure 5). Although HK1 cells are EBV , they were likely derived from EBV+ cells, in which certain cellular genes were irreversibly altered through genetic and epigenetic mechanisms.46 As a result, these alterations in HK1 cells might support an elevated activity of the EBER2 promoter. Nevertheless, the high activity of the EBER2 promoter in nasopharyngeal carcinoma cells can be utilized to deliver siRNA-based agents to study and combat this tumor. For example, the EBER2-shRNA expression vector might be particularly powerful in silencing EBV-specic genes, such as EBV nuclear antigen EBNA1, in EBV-infected tumor cells. EBNA1 is required for the maintenance of EBV episomes and the silencing of EBNA1 expression inhibits tumor cell growth.5961 Thus, the delivery of EBNA1-silencing
siRNAs via pEBER2-shRNA vector might prove useful in the development of new anti-EBV therapeutics.
While the biological functions of EBERs remain obscure and controversial, one of their potential functions is to promote cellular growth.62 We showed that the EBER sequences remaining in the shRNA fusion transcripts could neither activate PKR nor suppress RNAi (Figure 4), suggesting that these partial EBER sequences might not share the same PKR-modulatory properties as their full-length counterparts. However, it will still be of importance to clarify the inuence of these EBER sequences on cell growth and tumorigenesis before the EBER promoter-based vectors could be used to deliver any therapeutic agents into human cells.
Materials and methods
Construction of RNA expression vectors PCR and TA cloning were employed for the construction of shRNA expression vectors. The shRNA expression cassettes containing the promoters (that is, promoter-sense-loop-antisense-termination signal T6) were amplied by PCR. The EBER promoters were derived from EBV genomic DNA puried from B95-8 cells (GenBank accession number of EBV sequence: V01555; EBER sequences amplied: EBER1: 6510B6736, EBER2: 6856B7078). Human H1 promoter and human U6 promoter were PCR-subcloned from plasmids pSUPER (a gift from Reuven Agami, the Netherlands Cancer Institute)11 and pGEM-U6 (a gift from Greg Hannon, Cold Spring Harbor Laboratory).9 Human tRNAVal promoter was PCR-amplied from genomic DNA of HeLa cells. Expression vector for adenovirus VAI has been described.31 The EBER expression vector expresses both EBER1 and EBER2 under the control of their own promoters.
The shRNA against rey luciferase (shFluc) was derived from plasmid pSHAG-Ff1 (a gift from Greg Hannon).9 The shRNA targeting Rluc (shRluc) was targeted to 1776 to 1803 nucleotides of Rluc mRNA. The primers used were as follows: EBER1-shFluc, 50-GGA AAT GAG GGT TAG CAT AGG C-30 (forward) and 50-AAA AAA ATC AGG TGG CTC CCG CTG AAT TGG AAT CCC AAG CTT CGG ATT CCA ACT CAG CGA GAG CCA CCC GAT CTC GAG ATC GAT TCT AGA AGA CAA CCA CAG ACA CCG TC-30 (reverse); EBER2-shFluc, 50-GCT TAA CGT TGC ATC CCA GAA G-30 (forward) and 50- AAA AAA ATC AGG TGG CTC CCG CTG AAT TGG AAT CCC AAG CTT CGG ATT CCA ACT CAG CGA GAG CCA CCC GAT CTC GAG ATC GAT TCT AGA CTG ACT TGC AAA TGC TCT AGG C-30 (reverse); EBER1-shRluc, 50-GGA AAT GAG GGT TAG CAT AGG C-30 (forward) and 50- AAA AAA GTT AGG AAT TAT AAT GCT TAT CTA CGT GCC AAG CTT CGC ACA TAG ATA AGC ACT ATA ATC CCT AAC CTC GAG ATC GAT TCT AGA AGA CAA CCA CAG ACA CCG TC-30 (reverse); EBER2-shRluc, 50-GCT TAA CGT TGC ATC CCA GAA G-30 (forward) and 50-AAA AAA GTT AGG AAT TAT AAT GCT TAT CTA CGT GCC AAG CTT CGC ACA TAG ATA AGC ACT ATA ATC CCT AAC CTC GAG ATC GAT TCT AGA CTG ACT TGC AAA TGC TCT AGG C-30 (reverse); tRNAVal-shRluc, 50-TTG ATC CCG AAA GAT GTC CAG CG-30 (forward) and 50-AAA AAA GTT AGG AAT TAT AAT GCT TAT CTA CGT GCC AAG CTT CGC ACA TAG ATA AGC ACT ATA ATC CCT AAC CTC GAG ATC GAT TCT
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AGA TTT CCG CCC GGT TTC GAA C-30 (reverse); U6-shRluc, 50-ATT TAG GTG ACA CTA TAG-30 (forward) and 50-AAA AAA GTT AGG AAT TAT AAT GCT TAT CTA CGT GCC AAG CTT CGC ACA TAG ATA AGC ACT ATA ATC CCT AAC CTC GAG ATC GAT TCT AGA CGG TGT TTC GTC CTT TCC AC-30 (reverse); H1-shRluc, 50-TAA TAC GAC TCA CTA TAG GG-30 (forward) and 50-AAA AAA GTT AGG AAT TAT AAT GCT TAT CTA CGT GCC AAG CTT CGC ACA TAG ATA AGC ACT ATA ATC CCT AAC TCT AGA CTC GAG ATC GAT GGG AAA GAG TGG TCT CAT ACA GAA CTT ATA AGA TTC CC-30 (reverse).
EBER2-shRluc mutants were constructed using Quik-Change site-directed mutagenesis reagents (Stratagene, La Jolla, CA, USA). The shRNA sequences against Nup155 (shNup155) were designed with the help of the software provided by the Whitehead Institute (URL: http://jura.wi.mit.edu/bioc/siRNAext/
Web End =http://jura.wi.mit.edu/bioc/siRNAext/ ). The forward primer used to amplify EBER2-shNup155 was the same as that used for EBER2-shFluc. The reverse primers used were as follows: EBER2-shNup155 no. 1, 50-GAA GAT CTA AAA AAC ATG CAG GTG TTA GGT TAT ACA AGC TTC TAT AAC CTA ACA CCT GCA TGT GGA TCC CTG ACT TGC AAA TGC TCT AGG C-30; EBER2-shNup155 no. 2, GAA GAT CTA AAA AAG CCG GTT ATT CAG ACC CTA TAC AAG CTT CTA TAG GGT CTG AAT AAC CGG CGG ATC CCT GAC TTG CAA ATG CTC TAG GC-30. The forward primers used to PCR the shRNAs with scrambled sequence was those used for EBER2-shRluc and U6-shRluc. The reverse primers are as follows: EBER2-shSC, 50-AAA AAA GAA GAG AAC AGA ATA AGA GAT TCT CTT GAA ATC TCT TAT TCT GTT CTC TTC CTG ACT TGC AAA TGC TCT AGG C-30;
U6-shSC, 50-AAA AAA GAA GAG AAC AGA ATA AGA GAT TCT CTT GAA ATC TCT TAT TCT GTT CTC TTC GGT GTT TCG TCC TTT CCA C-30. The resulting products were then separately cloned either into pGEMT easy vector (Promega, Madison, WI, USA) or into pcDNA3.1 vector (Invitrogen, Carlsbad, CA, USA) with the CMV promoter removed.
Cell transfection and establishment of stable Nup155-knockdown cell linesHeLa, HEK293 and 293 T cells were cultured in Dulbeccos modied Eagles medium supplemented with 10% fetal bovine serum (FBS). C666-1 and HK1 cells were grown in RPMI1640 medium supplemented with 10% FBS. Cells were transfected at 50% conuence in 12-well plate, 6-well plate or 10 cm dish for 48 h using GeneJuice transfection reagent (Novagen, San Diego, CA, USA), FuGene 6 (Roche, Indianapolis, IN, USA) or Lipofectamine 2000 (Invitrogen). Plasmids expressing Fluc under the control of SV40 promoter (pGL3-Control) or Rluc driven by the CMV enhancer and early promoter (pRL-CMV) were from Promega.
To knockdown the expression of Fluc, pGL3-control, pRL-CMV and shRNA expression vector were cotransfected into cells in a ratio of 1:1:3. As such, cells in each well of the 12-well plate received 0.3 mg of pGL3-Control, 0.3 mg of pRL-CMV and 0.9 mg of shRNA vector. To target Rluc, the above vectors were used with either a ratio of 1:1:2 or 1:1:3. For assessing the specicity of the knockdown effect mediated by shRluc, pRL-CMV was replaced by pcDNA3.1/V5-His/lacZ (Invitrogen) in
the transfection. To evaluate vector inuence on PKR activity, 1 or 2 mg of shRNA expression vector was transfected. Plasmid pATF4-UTR-Luc used in the determination of eIF2a activation has been described.41
For the generation of stable Nup155-knockdown cell lines, HeLa cells were transfected with shRNA expression vectors carrying a G418 resistance gene cassette. After 48 h, the cells were selected with G418 (Calbiochem, San Diego, CA, USA) for 2 weeks before the isolation of single colonies.
Dual luciferase reporter assay Luciferase assays were performed as described.32,63,64
Briey, after rinsing with 1 PBS once, the harvested cells were lysed with 150 ml of passive lysis buffer (Promega) at room temperature for 15 min. Subsequently, 5 or 10 ml of the cell lysate was added to an opaque 96-well plate for measurement of luciferase activity using the Dual-Luciferase reporter assay system (Promega). The readouts of luciferase activity were taken in an LB 96 V microplate luminometer (EG&G, Victoria, Australia).
Luminescent b-galactosidase reporter assay Cells were harvested and lysed with passive lysis buffer. Measurement of b-galactosidase activity was performed using the luminescent b-galactosidase detection kit II (Clontech, Mountain View, CA, USA). The readouts of b-galactosidase activity were taken in an LB 96 V micro-plate luminometer (EG&G).
Northern blotting Total RNA was extracted from cells, 48 h after transfection, using Trizol reagent (Invitrogen). A total of 20 mg of total RNA was then separated by electrophoresis on a 12% polyacrylamide per 8 M urea gel and electroblotted onto a Zeta-Probe GT membrane (Bio-Rad, Hercules, CA, USA) for 1.5 h at 30 V. RNA was immobilized by UV crosslinking. Hybridization was carried out at 42 1C using Ultrahyb-Oligo hybridization buffer (Ambion, Austin, TX, USA) and a 32P-labeled Rluc sense DNA oligonucleotide. Membrane was washed twice for 20 min each at 42 1C with 2 SSC and 0.2% sodium dodecyl sulfate (SDS). Film was exposed overnight with an intensifying screen.
RNase protection assay Small RNAs (around 200 nucleotides or smaller) were extracted and enriched from transfected HEK293 in 10 cm dish using mirVana miRNA isolation kit (Ambion). A 32P-labeled Rluc sense RNA probe of 29 nucleotides in length was made using the mirVana miRNA probe construction kit (Ambion). Solution hybridization was carried out overnight at 42 1C with 10 mg of the enriched RNA and the gel-puried probe. After 24 h of hybridization, RNase treatment was performed for 1 h with 40 mgml 1 RNase A (USB) and 2 U of RNase T1 (Ambion). RNase was then inactivated with 5 ml of proteinase K (10 mg ml 1) in 10 ml of 20% SDS. The protected fragments were analyzed by polyacrylamide gel electrophoresis with 15% urea. Results were visualized using a phosphor imager (Molecular Dynamics, Sunnyvale, CA, USA).
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Quantitative RTPCR Total cellular RNA was extracted using Trizol and proteinase K. Briey, the cells were incubated with Trizol for 3 min at room temperature. The lysed samples were then treated with 10 ml of preheated proteinase K (10 mg ml 1) at 55 1C for 30 min. Total cellular RNA was extracted using the protocol provided by Invitrogen. After RNA extraction, 2 mg of the total cellular RNA was treated with 2 U of DNase (Ambion) at 37 1C for 15 min. DNase was heat-inactivated at 65 1C for 20 min. The synthesis of cDNA with oligo(dT)20 primer was then performed using the ThermoScript RTPCR system (Invitrogen). Subsequently, 2 ml of the synthesized cDNA was added to the quantitative PCR reaction mix containing 12.5 ml of 2 Brilliant SYBR Green QPCR master mix (Stratagene), 2.5 ml of forward primer (1 mM), 2.5 ml of reverse primer (1 mM) and 5.5 ml of water. Primers used to quantitate the Rluc mRNA were 50-ACG CTG AAA GTG TAG TAG A-30 (forward) and 50-AGA ATC CTG GGT CCG A-30 (reverse). For RNA normalization, quantitation of the Fluc mRNA was performed using 50-TCT ATC CGC TGG AAG ATG-30 (forward) and 50-ACT GTT GAG CAA TTC ACG-30 (reverse). Quantitative PCR was carried out in M 3000P real-time PCR system (Strata-gene) using the following thermal cycling prole: 1 cycle of 95 1C for 10 min, 40 cycles of amplication (95 1C for 30 s, 55 1C for 1 min and 72 1C for 30 s), followed by 41 cycles of incubation where the temperature increased by 1 1C per cycle beginning at 55 1C and ending at 95 1C. For each experimental setup, triplicate measurements were carried out and analyzed using the software (version1.20c) provided with the M 3000P system.
Antibodies and western blotting RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate) was used to lyse transfected HeLa or 293 T cells. To detect PKR and phosphorylated form of PKR, PhosphoStop solution (Roche) was also added to RIPA. The lysates were analyzed by SDSPAGE. The polyvinylidene uoride-membrane transferred with proteins was blocked with 5% skim milk in PBS and probed with primary antibodies. Primary antibodies used include rabbit polyclonal antibodies against PKR or phosphorylated form of PKR (Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal antibodies against Nup155 (kindly provided by Iain Mattaj, European Molecular Biology Laboratory),47 mouse monoclonal antibodies against b-actin (Sigma, St Louis, MO, USA) and mouse monoclonal antibodies against a- or b-tubulin (Sigma). Membranes were washed three times with Tris-buffered saline containing Tween 20 before donkey anti-rabbit or sheep anti-mouse HRP-conjugated secondary antibodies (GE Healthcare Life Sciences, Piscataway, NJ, USA) were added. After washing the membranes three times with TBST, bands were detected using the ECL system (GE Healthcare Life Sciences).
References
1 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.
Potent and specic genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391: 806811.2 Montgomery MK, Xu S, Fire A. RNA as a target of double-stranded RNA-mediated genetic interference in Caenorhabditis elegans. Proc Natl Acad Sci USA 1998; 95: 1550215507.3 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 2123 nucleotide intervals. Cell 2000; 101: 2533.4 Ghosh SK, Kusari J, Bandyopadhyay SK, Samanta H, Kumar R, Sen GC. Cloning, sequencing, expression of two murine 2050-oligoadenylate synthetases. Structure-function relationships. J Biol Chem 1991; 266: 1529315299.5 Samuel CE. The eIF-2a protein kinases, regulators of translation in eukaryotes from yeasts to humans. J Biol Chem 1993; 268: 76037606.6 Der SD, Yang YL, Weissmann C, Williams BR. A double-stranded RNA-activated protein kinase-dependent pathway mediating stress-induced apoptosis. Proc Natl Acad Sci USA 1997; 94: 32793283.7 Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, TuschlT. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 494498.8 Donze O, Picard D. RNA interference in mammalian cells using siRNAs synthesized with T7 RNA polymerase. Nucleic Acids Res 2002; 30: e46.9 Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specic silencing in mammalian cells. Genes Dev 2002; 16: 948958.10 Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 30 overhangs efciently suppress targeted gene expression in mammalian cells. Nat Biotechnol 2002; 20: 497500.11 Brummelkamp TR, Bernards R, Agami R. A system for stable expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550553.12 Lee NS, Dohjima T, Bauer G, Li H, Li MJ, Ehsani A et al. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002; 20: 500505.13 McManus MT, Petersen CP, Haines BB, Chen J, Sharp PA. Gene silencing using micro-RNA designed hairpins. RNA 2002; 8: 842850.14 Paul CP, Good PD, Winer I, Engelke DR. Effective expression of small interfering RNA in human cells. Nat Biotechnol 2002; 20: 505508.15 Sui G, Soohoo C, Affar EB, Gay F, Shi Y, Forrester WC et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 55155520.16 Yu JY, DeRuiter SL, Turner DL. RNA interference by expression of short-interfering RNAs and hairpin RNAs in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 60476052.17 Kim DH, Behlke MA, Rose SD, Chang MS, Choi S, Rossi JJ. Synthetic dsRNA Dicer substrates enhance RNAi potency and efcacy. Nat Biotechnol 2005; 23: 222226.18 Xia H, Mao Q, Paulson HL, Davidson BL. siRNA-mediated gene silencing in vitro and in vivo. Nat Biotechnol 2002; 20: 10061010.
Acknowledgements
We thank GJ Hannon, R Agami and IW Mattaj for reagents; KY Yuen for support with quantitative PCR analysis; C-P Chan, K-T Chin and K-L Siu for technical advice; and Y-P Ching, K-T Chin, ACS Chun and Y-T Siu
for critical reading of the manuscript. This work was supported by Innovation and Technology Fund of Hong Kong (ITS/112/02), Fogarty International Center of National Institutes of Health (R01 TW06186-01), Hong Kong Research Grants Council (HKU 1/06C), and University of Hong Kong (seed funding program for basic research 2004).
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19 Denti MA, Rosa A, Sthandier O, De Angelis FG, Bozzoni I. A new vector, based on the PolII promoter of the U1 snRNA gene, for the expression of siRNAs in mammalian cells. Mol Ther 2004; 10: 191199.
20 Schramm L, Hernandez N. Recruitment of RNA polymerase III to its target promoters. Genes Dev 2002; 16: 25932620.
21 Boden D, Pusch O, Lee F, Tucker L, Shank PR, Ramratnam B. Promoter choice affects the potency of HIV-1 specic RNA interference. Nucleic Acids Res 2003; 31: 50335038.
22 Kawasaki H, Taira K. Short hairpin type of dsRNAs that are controlled by tRNA(Val) promoter signicantly induce RNAi-mediated gene silencing in the cytoplasm of human cells. Nucleic Acids Res 2003; 31: 700707.
23 Pfeffer S, Zavolan M, Grasser FA, Chien M, Russo JJ, Ju J et al. Identication of virus-encoded microRNAs. Science 2004; 304: 734736.
24 Cai X, Schafer A, Lu S, Bilello JP, Desrosiers RC, Edwards R et al. Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog 2006; 2: e23.
25 Yuan J, Cahir-McFarland E, Zhao B, Kieff E. Virus and cell RNAs expressed during Epstein-Barr virus replication. J Virol 2006; 80: 25482565.
26 Thimmappaya B, Weinberger C, Schneider RJ, Shenk T. Adenovirus VAI RNA is required for efcient translation of viral mRNAs at late times after infection. Cell 1982; 31: 543551.
27 Howe JG, Shu MD. Epstein-Barr virus small RNA (EBER) genes: unique transcription units that combine RNA polymerase II and III promoter elements. Cell 1989; 57: 825834.
28 Howe JG, Shu MD. Upstream basal promoter element important for exclusive RNA polymerase III transcription of the EBER 2 gene. Mol Cell Biol 1993; 13: 26552665.
29 Wensing B, Stuhler A, Jenkins P, Hollyoake M, Karstegl CE, Farrell PJ. Variant chromatin structure of the oriP region of Epstein-Barr virus and regulation of EBER1 expression by upstream sequences and oriP. J Virol 2001; 75: 62356241.
30 Felton-Edkins ZA, Kondrashov A, Karali D, Fairley JA, Dawson CW, Arrand JR et al. Epstein-Barr virus induces cellular transcription factors to allow active expression of EBER genes by RNA polymerase III. J Biol Chem 2006; 281: 3387133880.
31 Kok KH, Jin DY. Inuenza A virus NS1 protein does not suppress RNA interference in mammalian cells. J Gen Virol 2006; 87: 26392644.
32 Kok KH, Ng MHJ, Ching YP, Jin DY. Human TRBP and PACT directly interact with each other and associate with Dicer to facilitate the production of small interfering RNA. J Biol Chem 2007; 282: 1764917657.
33 Toczyski DP, Matera AG, Ward DC, Steitz JA. The Epstein-Barr virus (EBV) small RNA EBER 1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes. Proc Natl Acad Sci USA 1994; 91: 34633467.
34 Elia A, Laing KG, Schoeld A, Tilleray VJ, Clemens MJ. Regulation of the double-stranded RNA-dependent protein kinase PKR by RNAs encoded by a repeated sequence in the Epstein-Barr virus genome. Nucleic Acids Res 1996; 24: 44714478.
35 Fok V, Friend K, Steitz JA. Epstein-Barr virus noncoding RNAs are conned to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling. J Cell Biol 2006; 173: 319325.
36 Nanbo A, Yoshiyama H, Takada K. Epstein-Barr virus-encoded poly(A)-RNA confers resistance to apoptosis mediated through Fas by blocking the PKR pathway in human epithelial intestine 407 cells. J Virol 2005; 79: 1228012285.
37 Ruf IK, Lackey KA, Warudkar S, Sample JT. Protection from interferon-induced apoptosis by Epstein-Barr virus small RNAs is not mediated by inhibition of PKR. J Virol 2005; 79: 14562 14569.
38 Wong HL, Wang X, Cheng RCC, Jin DY, Feng H, Wang Q et al. Stable expression of EBERs in immortalized nasopharyngeal
epithelial cells confers resistance to apoptotic stress. Mol Carcinog 2005; 44: 92101.39 Lu PD, Harding HP, Ron D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J Cell Biol 2004; 167: 2733.40 Vattem KM, Wek RC. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci USA 2004; 101: 1126911274.41 Chan CP, Siu KL, Chin KT, Yuen KY, Zheng B, Jin DY. Modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein. J Virol 2006; 80: 92799287.42 Glickman JN, Howe JG, Steitz JA. Structural analyses of EBER1 and EBER2 ribonucleoprotein particles present in Epstein-Barr virus-infected cells. J Virol 1988; 62: 902911.43 Aparicio O, Razquin N, Zaratiegui M, Narvaiza I, Fortes P. Adenovirus virus-associated RNA is processed to functional interfering RNAs involved in virus production. J Virol 2006; 80: 13761384.44 Bennasser Y, Yeung ML, Jeang KT. HIV-1 TAR RNA subverts RNA interference in transfected cells through sequestration of TAR RNA binding protein, TRBP. J Biol Chem 2006; 281: 2767427678.45 Cheung ST, Huang DP, Hui AB, Lo KW, Ko CW, Tsang YS et al. Nasopharyngeal carcinoma cell line (C666-1) consistently harbouring Epstein-Barr virus. Int J Cancer 1999; 83: 121126.46 Huang DP, Ho JH, Poon YF, Chew EC, Saw D, Lui M et al. Establishment of a cell line (NPC/HK1) from a differentiated squamous carcinoma of the nasopharynx. Int J Cancer 1980; 26: 127132.47 Franz C, Askjaer P, Antonin W, Iglesias CL, Haselmann U, Schelder M et al. Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates. EMBO J 2005; 24: 35193531.48 Dumpelmann E, Mittendorf H, Benecke BJ. Efcient transcription of the EBER2 gene depends on the structural integrity of the RNA. RNA 2003; 9: 432442.49 Howe JG, Steitz JA. Localization of Epstein-Barr virus-encoded small RNAs by in situ hybridization. Proc Natl Acad Sci USA 1986; 83: 90069010.50 Paul CP, Good PD, Li SX, Kleihauer A, Rossi JJ, Engelke DR. Localized expression of small RNA inhibitors in human cells. Mol Ther 2003; 7: 237247.51 Robb GB, Brown KM, Khurana J, Rana TM. Specic and potent RNAi in the nucleus of human cells. Nat Struct Mol Biol 2005; 12: 133137.52 Berezhna SY, Supekova L, Supek F, Schultz PG, Deniz AA. siRNA in human cells selectively localizes to target RNA sites. Proc Natl Acad Sci USA 2006; 103: 76827687.53 Morris KV, Chan SW, Jacobsen SE, Looney DJ. Small interfering RNA-induced transcriptional gene silencing in human cells. Science 2004; 305: 12891292.54 Weinberg MS, Villeneuve LM, Ehsani A, Amarzguioui M, Aagaard L, Chen Z-X et al. The antisense strand of small interfering RNAs direct histone methylation and transcriptional gene silencing in human cells. RNA 2005; 12: 256262.55 Ting AH, Schuebel KE, Herman JG, Baylin SB. Short double-stranded RNA induces transcriptional gene silencing in human cancer cells in the absence of DNA methylation. Nat Genet 2005; 37: 906910.56 Tao Q, Young LS, Woodman CB, Murray PG. Epstein-Barr virus (EBV) and its associated human cancers-genetics, epigenetics, pathobiology and novel therapeutics. Front Biosci 2006; 11: 26722713.57 Klein E, Kis LL, Klein G. Epstein-Barr virus infection in humans: from harmless to life endangering virus-lymphocyte interactions. Oncogene 2007; 26: 12971305.
201
Gene Therapy
Induction of RNAi using EBER-shRNA transcripts EY-W Choy et al
202
58 Wang Y, Xue SA, Hallden G, Francis J, Yuan M, Grifn BE et al. Virus-associated RNA I-deleted adenovirus, a potential oncolytic agent targeting EBV-associated tumors. Cancer Res 2005; 65: 15231531.
59 Nasimuzzaman M, Kuroda M, Dohno S, Yamamoto T, Iwatsuki K, Matsuzaki S et al. Eradication of Epstein-Barr virus episome and associated inhibition of infected tumor cell growth by adenovirus vector-mediated transduction of dominant-negative EBNA1. Mol Ther 2005; 11: 578590.
60 Hong M, Murai Y, Kutsuna T, Takahashi H, Nomoto K, Cheng CM et al. Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitts lymphoma cells. J Cancer Res Clin Oncol 2006; 132: 18.
61 Yin Q, Flemington EK. siRNAs against the Epstein Barr virus latency replication factor, EBNA1, inhibit its function and growth of EBV-dependent tumor cells. Virology 2006; 346: 385393.
62 Nanbo A, Takada K. The role of Epstein-Barr virus-encoded small RNAs (EBERs) in oncogenesis. Rev Med Virol 2002; 12: 321326.
63 Zhou Y, Ching YP, Kok KH, Kung HF, Jin DY. Post-transcriptional suppression of gene expression in Xenopus embryos by small interfering RNA. Nucleic Acids Res 2002; 30: 16641669.
64 Siu YT, Chin KT, Siu KL, Choy EYW, Jeang KT, Jin DY. TORC1 and TORC2 coactivators are required for Tax activation of the human T-cell leukemia virus type 1 long terminal repeats. J Virol 2006; 80: 70527059.
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