Direct detection of RNA transcription by FRET imaging using fluorescent protein probe

Available online at www.sciencedirect.com

Journal of Biotechnology 133 (2008) 413–417

Direct detection of RNA transcription by FRET imaging using fluorescent protein probe Tamaki Endoh, Masayasu Mie, Eiry Kobatake ∗ Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan Received 10 September 2007; accepted 5 November 2007

Abstract We have constructed a reporter system for intracellular direct detection of RNA transcription that consists of two biomolecular components. The first part is a GFP-based recombinant protein probe (YRG0C-11ad) containing the RNA-binding Rev-peptide between ECFP and EYFP. The second component is RRE-RNA, which specifically binds to the Rev-peptide. Cells stably expressing YRG0C-11ad were identified by an increased FRET signal after direct transfection or intracellular transcription of RRE-RNA. In addition, the signal increase is more noticeable if tandemly repeated RRE-RNA is used as the reporter. Untranslatable non-coding RNAs are regarded as regulators of cellular gene expression, but they are difficult to study using indirect reporter systems that are dependent on translational products. Direct detection of reporter RNA would be a useful method for the detection of intracellular promoter activity during transcription of untranslatable RNAs. © 2007 Elsevier B.V. All rights reserved. Keywords: Fluorescence resonance energy transfer (FRET); Peptide–RNA interaction; HIV-1 Rev-peptide; Rev response element (RRE)-RNA; RNA imaging

1. Introduction Recent transcriptomic analyses have revealed the presence of a great variety of untranslatable non-coding RNAs (Cawley et al., 2004; Mattick, 2001). The transcriptional levels of these RNAs indicate varied patterns during embryogenesis and cellular differentiation (Costa, 2005; Mattick and Makunin, 2005). They are drawing attention as key biomolecules in the establishment of highly organized cellular functions and biological mechanisms. It is possible that they contribute to the regulation of gene expression at both the transcriptional and post-transcriptional levels (Mattick, 2004). Although previous research efforts have clarified functional mechanisms of the gene expression control, the transcriptional control of non-coding RNA itself is still not well characterized. Several reporter assay systems have been developed to detect and monitor intracellular biological events. The output signals in most of these systems result from the expression of reporter proteins, such as GFP, luciferase, and ␤-galactosidase (de Wet et al.,

∗

Corresponding author. Tel.: +81 45 924 5760; fax: +81 45 924 5779. E-mail address: [email protected] (E. Kobatake).

0168-1656/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2007.11.005

1985; Naylor, 1999; Prasher, 1995; Prasher et al., 1992). One of the most typical assays used by these reporter systems is to monitor the activation of transcriptional factors responding to signal transductions (Alam and Cook, 1990; Naylor, 1999). Reporter proteins are expressed from transfected vectors, mediated by the binding of transcriptional factors to a corresponding promoter element inserted upstream of the reporter protein (Chen et al., 1995; Sista et al., 1994). However, this is an indirect assay of transcriptional factor activity because the reporter signal is emitted only after translation of the mRNA. There will always be a time-lag between actual transcription and signal observation (Imai et al., 2004). In addition, it cannot be used to determine what biological factors cooperate with RNA polymerases (e.g., RNA polymerase III and viral RNA polymerase) that transcribe untranslatable RNA (Goodfellow et al., 2006; Jakubiec et al., 2006; Larminie et al., 1997; Piccininni et al., 2002). Therefore, a reporter system that allows intracellular direct detection of RNA transcription, without the requirement of translational processes, is needed. In a previous report, we demonstrated the detection of a specific nucleic acid sequence using a fluorescent protein probe and split-RNA probe sets (Endoh et al., 2005). HIV-1 Revpeptide was inserted between ECFP and EYFP, a FRET donor

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fragment was obtained from previously constructed plasmid pCMV-YRG0C-11ad (Endoh et al., 2005). IRES element was obtained from pIRES2-EGFP (Clontech). And BSD gene fragment was obtained from pMAM2-BSD (Kaken Pharmaceutical). Hybridized DNA oligonucleotides coding RRE-RNA and BS-RNA sequence were inserted between BamHI and HindIII site of pBAsi-hU6 Pur (Takara) to construct plasmid vectors for reporter RNA transcription. DNA oligonucleotides for RRERNA are 5 -GATCCGGGTCTGGGCGCAGCGCAAGCTGACGGTACAGGCCCTTTTTTA-3 and 5 -AGCTTAAAAAAGGGCCTGTACCGTCAGCTTGCGCTGCGCCCAGACCCG3 , and for BS-RNA are 5 -GATCCGGGCGAATTGGGTACCGGGCCCCCCCTCGAGGTCTTTTTTA-3 and AGCTTAAAAAAGACCTCGAGGGGGGGCCCGGTACCCAATTCGCCCG-3 . Plasmid vectors for tandemly repeated reporter RNA transcription, RRE-RNA and TAR-RNA, were constructed as the same way. DNA oligonucleotides for RRE-RNA repeat are 5 -GATCCGGCCTGGGCGCAGCGCAAGCTGACGGTACAGGCCAAACCGGAAAAGATCTTTTTTTA-3 and 5 -AGCTTAAAAAAAGATCTTTTCCGGTTTGGCCTGTACCGTCAGCTTGCGCTGCGCCCAGGCCG-3 , and for TAR-RNA repeat are 5 -GATCCGGCTCGTGTAGCTCATTAGCTCCGAGCCAAACCGGAAAAGATCTTTTTTTA-3 and 5 -AGCTTAAAAAAAGATCTTTTCCGGTTTGGCTCGGAGCTAATGAGCTACACGAGCCG-3 . Repeated fragments of the reporter RNA (×4 and ×16) were constructed by ligation between BamHI and BglII sites in both sides of the inserted DNA oligonucleotide. Sequences of the transcriptional products are shown in Fig. 1b. Fig. 1. (a) Principle of intracellular direct detection of reporter RNA. The reporter RNA transcribed from the promoter binds to the protein probe followed by a change of FRET signal. (b) Structure of the constructed plasmid vectors. RNA sequences of the transcriptional products are shown on the respective images.

2.2. Cell culture and RNA preparation

and acceptor pair. The constructed fluorescent protein probe (YRG0C-11ad) displayed a FRET signal increase or decrease mediated by the conformational change of the inserted Revpeptide after specific binding of RRE-RNA or Rev-aptamer. In this system, RRE-RNA and Rev-aptamer act as reporter RNAs, which are directly detected by the change in FRET signal intensity. Here, we tested the intracellular detection of the reporter RNA using cells expressing YRG0C-11ad protein probe (Fig. 1a).

HeLa cells were seeded at 0.5 × 105 cells per well on a 6well plate and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin (50 u/mL), and streptomycin (50 ␮g/mL). After a day’s culture, the cells were transfected with 2.0 ␮g pEF1␣YRG0C-11ad-IRES-BSD by using 3.0 ␮L FuGENE6 (Roche). Transfected cells were continuously cultured in DMEM containing 10 ␮g/mL blasticidin S to establish stably transfected cell line. RNA oligonucleotides (RRE-RNA, Rev-aptamer, and BSRNA) were synthesized from DNA template and purified according to previous report (Endoh et al., 2005).

2. Materials and methods

2.3. Intracellular FRET imaging

2.1. Plasmid construction

Stably transfected cell line expressing YRG0C-11ad was seeded on a glass base dish (Iwaki) and grown to 70% confluence in DMEM without blasticidin S. Synthesized RNA (320 pmol) or vectors for RNA transcription (2.0 ␮g) were transfected into the cells using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. After 6 h from direct RNA transfection or 24 h from vector transfection, culture medium was exchanged to HEPES buffer (20 mM HEPES-KOH (pH 7.4), 115 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2 , 0.8 mM MgCl2

Gene fragments, YRG0C-11ad and blasticidin S deaminase (BSD), and DNA element, EF1-␣ promoter and internal ribosomal entry site (IRES), were connected and inserted into luciferase deleted Pica Gene Basic Vector 2 (Toyo Inc.) to construct a pEF1␣-YRG0C-11ad-IRES-BSD for the expression of the protein probe. EF1-␣ promoter element was obtained from pEF5/FRT/V5/D-TOPO (Invitrogen). YRG0C-11ad gene

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and 13.8 mM glucose) following two times washing. Cellular FRET image, the ratio of EYFP/ECFP fluorescence intensity, was obtained using AquaCosmos/FRET W-view (Hamamatsu Photonics). Averaged FRET signal intensity was calculated from images obtained from different areas. Both fluorescence intensity, EYFP and ECFP, were normalized by subtracting the intensity of glass base dish without cells. 3. Results and discussion 3.1. Intracellular FRET signal change after direct RNA transfection A HeLa cell line has been established that expresses the YRG0C-11ad fluorescent protein probe. A strong cytomegalovirus (CMV) promoter was utilized to express a sufficient amount of the protein probe for in vitro experiments (Endoh et al., 2005). The amount of excess protein probe was expected to lower the signal–noise ratio (S/N), because the signal change after reporter RNA binding would be indistinguishable behind the signals from unbound protein probes. Therefore, we constructed a new plasmid containing the EF1-␣ promoter instead of the CMV promoter. A cell line indicating minimal fluorescence intensity for FRET imaging was established using blasticidin S. The intracellular function of the YRG0C-11ad protein probe was evaluated by direct transfection of synthesized RNA oligonucleotides into the established cell line. Cellular FRET images were obtained 6 h after transfection (Fig. 2a–c). The majority of RRE-RNA-transfected cells exhibited a higher FRET signal compared to the non-treated cell line. In contrast, cells transfected with BS-RNA, the control RNA oligonucleotide, showed no changes in FRET signal intensity. We also evaluated averaged FRET signal values from cellular FRET images (Fig. 2d). The range of FRET increase after RRE-RNA transfection corresponded well to the in vitro character of the YRG0C-11ad protein probe. In contrast, the FRET signal in Rev-aptamer-transfected cells was indistinguishable from that of control RNA-transfected cells and non-treated cells, although FRET decrease was previously confirmed in vitro. It is possible that the Rev-aptamer has a weaker interaction affinity than RRERNA for intracellular YRG0C-11ad. These results confirmed the intracellular function of the YRG0C-11ad protein probe and the capacity to detect the intracellular RRE-RNA by FRET signal increase. Therefore, RRE-RNA was chosen to assay for direct detection of intracellular RNA transcription. 3.2. Intracellular FRET signal change after RNA transcription To evaluate whether intracellular RNA transcription can be directly detected by a FRET signal change, plasmid vectors for RNA transcription were transfected into the YRG0C-11ad cell line. The human U6 promoter is found in the promoter region of human U6 small nuclear RNA that is included in spliceosomes (Kunkel et al., 1986). It is suitable for the transcription of short RNAs, such as short hairpin RNA (shRNA) for RNAi,

Fig. 2. (a–c) Cellular FRET image after direct RNA transfection. Synthesized RNAs, BS-RNA (a), RRE-RNA (b), Rev-aptamer (c), were transfected using lipofectamine transfection reagent. FRET images were obtained after 6 h incubation. (d) Averaged FRET signal intensity (EYFP/ECFP) was culculated from different areas (n = 7).

because it is recognized and transcribed by RNA polymerase III (Lee et al., 2002; Tuschl, 2002). In addition, the transcribed RNA is not translated by ribosomes because it does not have an inverted 7-meG triphosphate (cap) at the 5 end. The human U6 promoter was chosen as a model for intracellular transcription of untranslatable RNAs, and was used to transcribe RRE-RNA. YRG0C-11ad Hela cells were transfected with either pBAsihU6-RRE (for transcription of RRE-RNA) or the negative control vector pBAsi-hU6-BS (for transcription of BS-RNA). Cellular FRET images were obtained 24 h after vector transfection (Fig. 3a–c). Some of the cells transfected with pBAsi-hU6-RRE produced higher FRET signals compared to

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3.3. FRET signal changes after tandemly repeated reporter RNA transcription Although intracellular reporter RNA transcription was detected by an increase in FRET signaling, the intensity of FRET signal increase was smaller than in the case of direct RNA transfection. We speculated that the copy number of RRERNA transcribed from U6 promoter was lower than the number of the YRG0C-11ad protein probes expressed in the cells, leading to a surplus of unbound protein probes. To test this theory, we constructed a plasmid vector that transcribes an RRE-RNA tandem repeat to improve the FRET intensity. A plasmid vector that transcribes a tandemly repeated TAR-RNA, the RNA partner of a well-characterized protein–RNA (Tat-TAR) interaction, was prepared as a negative control. Fig. 4a shows the averaged FRET signal values 24 h after transfection. Cells with tandem repeats of RRE-RNA had higher FRET signals than those with tandemly repeated TAR-RNA. The more RRE-RNA repeats, the higher the FRET signal, suggesting that the signal was ampli-

Fig. 3. (a–c) Cellular FRET signal increase respond to reporter RNA transcription. Plasmid vectors, pBAsi-hU6-BS (b) or pBAsi-hU6-RRE (c), were transfected using lipofectamine transfection reagent. FRET images were obtained after 24 h incubation. (d) Averaged FRET signal intensity (EYFP/ECFP) was calculated from different areas (n = 4).

circumjacent cells. It is possible that those cells with higher FRET signals were more efficiently transfected with the plasmid vector and transcribed a sufficient amount of RRE-RNA for a FRET signal increase. In contrast, negative control cells transfected with pBAsi-hU6-BS had FRET signals comparable to those of non-transfected cells. These results indicate that it is possible to detect intracellular reporter RNA transcription using FRET signal imaging. However, when FRET signal values were averaged from all of the cellular images, the difference in signal intensity was obscured because of the relatively large error bars (Fig. 3d). Therefore, the difference was considered small due to an influence of lower FRET signals from the cells not efficiently transfected with plasmid vector. This observation suggests a superiority of direct FRET imaging in distinguishing subtle differences.

Fig. 4. (a) Averaged FRET signal intensity (EYFP/ECFP) after transcription of tandemly repeated reporter RNA. Plasmid vectors were transfected using lipofectamine transfection reagent. FRET signal ratio was calculated from different areas after 24 h incubation (n = 7). (b–d) Cellular FRET image of transfected cells, pBAsi-hU6-Pur (b), pBAsi-hU6-tTAR × 16 (c), pBAsi-hU6-tRRE × 16 (d).

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fied by the binding of several YRG0C-11ad protein probes to one copy of reporter RNA. Cells transfected with pBAsi-hU6tRRE × 16 (Fig. 4d) obviously emitted higher FRET signals than control cells transfected with pBAsi-hU6 Pur (Fig. 4b) or pBAsi-hU6-tTAR × 16 (Fig. 4d). 4. Conclusion We demonstrated successful direct detection of intracellular reporter RNA transcription using the YRG0C-11ad protein probe. Cellular FRET signal was increased after transcription of RRE-RNA under the human U6 promoter, which transcribes untranslatable RNA through an RNA polymerase III. In addition, the increase in FRET signal was amplified by the transcription of tandemly repeated reporter RNA, which enables the detection of fewer copies of RNA transcripts. Although there are several reports that provide details of RNA transcript detection in living cells using oligonucleotide probes such as molecular beacons (Bratu et al., 2003; Fang et al., 2002; Tyagi and Kramer, 1996), our method consists of a recombinant protein probe and reporter RNA. FRET signal output can be observed continuously, since there is no need to introduce probes into cells once the protein probe is stably transformed. This system would be useful in the analysis of biological factors that contribute to RNA transcriptional activity, especially those that regulate untranslatable RNA transcripts. Acknowledgement This study was in part supported by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. References Alam, J., Cook, J.L., 1990. Reporter genes: application to the study of mammalian gene transcription. Anal. Biochem. 188, 245–254. Bratu, D.P., Cha, B.J., Mhlanga, M.M., Kramer, F.R., Tyagi, S., 2003. Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl. Acad. Sci. U.S.A. 100, 13308–13313. Cawley, S., Bekiranov, S., Ng, H.H., Kapranov, P., Sekinger, E.A., Kampa, D., Piccolboni, A., Sementchenko, V., Cheng, J., Williams, A.J., Wheeler, R., Wong, B., Drenkow, J., Yamanaka, M., Patel, S., Brubaker, S., Tammana, H., Helt, G., Struhl, K., Gingeras, T.R., 2004. Unbiased mapping of transcription factor binding sites along human chromosomes 21 and 22 points to widespread regulation of noncoding RNAs. Cell 116, 499–509. Chen, W., Shields, T.S., Stork, P.J., Cone, R.D., 1995. A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal. Biochem. 226, 349–354.

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Costa, F.F., 2005. Non-coding RNAs: new players in eukaryotic biology. Gene 357, 83–94. de Wet, J.R., Wood, K.V., Helinski, D.R., DeLuca, M., 1985. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82, 7870–7873. Endoh, T., Funabashi, H., Mie, M., Kobatake, E., 2005. Method for detection of specific nucleic acids by recombinant protein with fluorescent resonance energy transfer. Anal. Chem. 77, 4308–4314. Fang, X., Mi, Y., Li, J.J., Beck, T., Schuster, S., Tan, W., 2002. Molecular beacons: fluorogenic probes for living cell study. Cell. Biochem. Biophys. 37, 71–81. Goodfellow, S.J., Innes, F., Derblay, L.E., MacLellan, W.R., Scott, P.H., White, R.J., 2006. Regulation of RNA polymerase III transcription during hypertrophic growth. EMBO J. 25, 1522–1533. Imai, K., Nishiwaki, T., Kondo, T., Iwasaki, H., 2004. Circadian rhythms in the synthesis and degradation of a master clock protein KaiC in cyanobacteria. J. Biol. Chem. 279, 36534–36539. Jakubiec, A., Tournier, V., Drugeon, G., Pflieger, S., Camborde, L., Vinh, J., Hericourt, F., Redeker, V., Jupin, I., 2006. Phosphorylation of viral RNAdependent RNA polymerase and its role in replication of a plus-strand RNA virus. J. Biol. Chem. 281, 21236–21249. Kunkel, G.R., Maser, R.L., Calvet, J.P., Pederson, T., 1986. U6 small nuclear RNA is transcribed by RNA polymerase III. Proc. Natl. Acad. Sci. U.S.A. 83, 8575–8579. Larminie, C.G., Cairns, C.A., Mital, R., Martin, K., Kouzarides, T., Jackson, S.P., White, R.J., 1997. Mechanistic analysis of RNA polymerase III regulation by the retinoblastoma protein. EMBO J. 16, 2061–2071. Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M.J., Ehsani, A., Salvaterra, P., Rossi, J., 2002. Expression of small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nat. Biotechnol. 20, 500– 505. Mattick, J.S., 2001. Non-coding RNAs: the architects of eukaryotic complexity. EMBO Rep. 2, 986–991. Mattick, J.S., 2004. RNA regulation: a new genetics? Nat. Rev. Genet. 5, 316–323. Mattick, J.S., Makunin, I.V., 2005. Small regulatory RNAs in mammals. Hum. Mol. Genet. 14, R121–R132 (Spec no. 1). Naylor, L.H., 1999. Reporter gene technology: the future looks bright. Biochem. Pharmacol. 58, 749–757. Piccininni, S., Varaklioti, A., Nardelli, M., Dave, B., Raney, K.D., McCarthy, J.E., 2002. Modulation of the hepatitis C virus RNAdependent RNA polymerase activity by the non-structural (NS) 3 helicase and the NS4B membrane protein. J. Biol. Chem. 277, 45670– 45679. Prasher, D.C., 1995. Using GFP to see the light. Trends Genet. 11, 320–323. Prasher, D.C., Eckenrode, V.K., Ward, W.W., Prendergast, F.G., Cormier, M.J., 1992. Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111, 229–233. Sista, P., Edmiston, S., Darges, J.W., Robinson, S., Burns, D.J., 1994. A cellbased reporter assay for the identification of protein kinase C activators and inhibitors. Mol. Cell. Biochem. 141, 129–134. Tuschl, T., 2002. Expanding small RNA interference. Nat. Biotechnol. 20, 446–448. Tyagi, S., Kramer, F.R., 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14, 303–308.