Duration Control of Protein Expression in Vivo by Light-Mediated

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Duration Control of Protein Expression in Vivo by Light-Mediated Reversible Activation of Translation Shinzi Ogasawara* Creative Research Institution Sousei, Hokkaido University, Kita 21 Nishi 10, Kita-ku, Sapporo, Hokkaido 001-0021, Japan PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan S Supporting Information *

ABSTRACT: The photocontrol of protein expression enables the spatiotemporal induction of biological events in living cells or organisms. However, commonly used method such as photocontrollable transcription factor or caged nucleic acids is unsuitable for precise control of the duration of protein expression. Here, I report a photocontrollable cap (PC-cap) that can control the translation of mRNA in a reversible manner via its cis−trans photoisomerization through illumination with 370 and 430 nm light. 2-meta-Methyl-phenylazo cap (mMe-2PA-cap) in the trans form silences translation in zebrafish embryo, whereas treatment with the cis form provided a 7.1 times larger amount of translated protein compared to the trans form. Moreover, translation activated by illumination of the embryo with 370 nm light was rapidly inactivated again by subsequent illumination with 430 nm light. An application of this approach was demonstrated by photoinducing the development of double-headed zebrafish by controlling the expression period of squint protein.

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directly regulate the translation of mRNA. mRNA-based translational control has additional advantages, including nonincorporation into the host genome and complete degradation within a few days when used in medical applications. Caged nucleic acids such as caged antisense16 and mRNA18 are commonly used strategies for the photoregulation of translation. However, these methods allow for only a single off-to-on or on-to-off regulation event because the uncaged nucleic acid produced upon light illumination cannot be recaged, thus preventing control of the duration of protein expression. We previously demonstrated the potency of the strategy for controlling translation in a reversible manner by photoregulating the interaction between cap and eIF4E.20,21 However, the photregulation requires ultraviolet B that can critically damage biomolecules and ultimately cause necrosis. For example, development of the zebrafish embryo is stopped in midblastula transition and broken within several hours by illumination with 310 nm light (Figure S5). This is probably due to damage of the DNA by 310 nm light illumination. Additional efforts are thus needed to create more broadly applicable methods promising reversible control of translation in vivo. Here, I report a PC-cap that can reversibly control translation by illumination with 370 and 430 nm light and demonstrate the application of this approach by photoinducing the complete secondary axis in the zebrafish embryo by controlling the expression period of squint protein.

uration of protein expression is important factor for biological events, especially for embryonic development. For example, squint is expressed only during the period between the eight-cell stage and the shield stage in zebrafish embryo and leads to a normal dorsal−ventral axis.1,2 However, a prolonged overexpression system is typically used to investigate the protein function. Alternative strategies are to control transcription3−13 or translation14−18 with light, thus allowing easy control of the location and time of protein expression. A transcriptional control method based on a fused protein consisting of transcription factor Gal4(65) and vivid (VVD), the smallest light-oxygen-voltage domain, is one of the effective methods reported to date.5 Under blue light illumination, a Gal4(65)−VVD fusion protein dimerizes and binds to the upstream activator sequence of a specific gene and activates transcription. In the absence of illumination, the dimer gradually dissociates with a half-life of 2 h and finally inactivates transcription. This system exhibits a long response time lag for regulation. Several hours elapse between illumination with blue light and the beginning of protein expression, and protein continues to be synthesized for more than 10 h after turning the light off due to residual mRNA and slow dissociation of the dimer. Therefore, this method is unsuitable for precise control of the protein expression period. Furthermore, transcription does not occur until the midblastula transition, about 3.5 h following fertilization, whereas translation begins soon after fertilization in the zebrafish embryo. Therefore, cell fate is determined only by translation of maternal mRNA, including squint mRNA, in the early developmental stage.19 The control of embryonic development thus requires methods that can © XXXX American Chemical Society

Received: August 9, 2016 Accepted: December 15, 2016

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DOI: 10.1021/acschembio.6b00684 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology Scheme 1. Synthesis of PC-capsa

a Reagents and conditions: (a) ArNH2, AcOH, anhydrous acetonitrile, RT, 3 h. (b) Sodium methylate, anhydrous methanol, RT, 3 h. (c) Proton sponge, POCl3, (OMe)3P, 0 °C, 16 h. (d) NaNO2, AcOH, water, RT, 20 h. (e) Dimethyl sulfate, water, RT, 4 h. (f) GDP imidazolide, ZnCl2, anhydrous DMF, RT, 72 h.

360 and 460 nm at 10 nm intervals by illuminating with monochromic light. The most skewed trans/cis ratios of 2PAcap, pMe-2PA-cap, and mMe-2PA-cap were 23:77, 6:94, and 21:79 at 370 nm and 82:18, 74:26, and 82:18 at 430 nm, respectively (Figure S1). These results indicate that highly reversible trans−cis photoswitching of PC-caps can be realized by alternate illumination with 370 and 430 nm light. In both trans-to-cis and cis-to-trans photoisomerization, the photostationary state was reached within 2 min using 370 or 430 nm light illumination (Figures 1b, S2b, and S3b). Reversible switching was repeated 40 times by alternate illumination with 370 and 430 nm light, and good reversibility of trans−cis photoisomerization was observed without any side reactions (Figures 1c, S2c, and S3c). The thermal cis-to-trans isomerization rate was measured at 28.5 °C. Figure 1d shows the time profile of transient absorbance for mMe-2PA-cap monitored at 323 nm after excitation with 370 nm light for 2 min. The thermal cis-to-trans isomerizations of 2PA-cap, pMe-2PA-cap, and mMe-2PA-cap were observed with time constants of 2.42, 0.73 and 1.30 h, respectively. Introduction of the PC-caps to the 5′-end of the mRNA was accomplished by in vitro transcription. The efficiency in capping process of PC-caps was the same as that of normal-cap.

Introduction of a phenylazo group into the C8 position of guanosine induced a thermally unstable cis isomer.22 Therefore, I attempted to modify C2 amine located on the opposite side of the C8 position. Three PC-caps, 2-phenylazo cap (2PA-cap), 2para-methyl-phenylazo cap (pMe-2PA-cap), and 2-meta-methyl-phenylazo cap (mMe-2PA-cap), were designed to modulate the affinity for eIF4E by the difference in the steric hindrance. The PC-caps were synthesized according to Scheme 1. The structures of the PC-caps were characterized by 1H, 13C, and 31 P NMR and mass spectroscopy (Supporting Information). Introduction of the PC-cap into the 5′-end of the target mRNA was accomplished by in vitro transcription. Photoisomerization of PC-caps was demonstrated in aqueous solution using a 300 W xenon lamp. The photoisomerization properties of the mMe-2PA-cap are summarized in Figure 1 and in the Supporting Information for 2PA-cap and pMe-2PA-cap. The absorption spectra of the trans forms showed a peak at 310, 331, and 323 nm for 2PA-cap, pMe-2PA-cap, and mMe-2PAcap, respectively, corresponding to ππ* absorption (Figures 1a, S2a, and S3a), whereas the absorption maxima of the cis forms were at 416, 418, and 417 nm, respectively, corresponding to the nπ* absorption. The wavelength-dependence of the trans/ cis ratio in the photostationary state was investigated between B

DOI: 10.1021/acschembio.6b00684 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 1. Photoisomerization properties of mMe-2PA-cap. (a) Absorption spectra of the trans and cis isomer. (b) Time course of trans-to-cis and cisto-trans photoisomerization. (c) Switching cycles between trans and cis by alternate illumination with 370 and 430 nm light for 2 min. (d) Time profile of thermal cis-to-trans isomerization at 28.5 °C. The transient absorbance monitored at 323 nm was converted to trans-isomer percentage.

2PA-cap was in the trans-form due to illumination with 430 nm light before microinjection. Fluorescence gradually increased between 3 hpf and 7 hpf by illumination with 370 nm light every 1 h, indicating that the mRNA was translated. Subsequent illumination with 430 nm light at 7 hpf resulted in a slow rate of increase in fluorescence, suggesting that translation was inhibited again. Finally, to demonstrate the applicability of my method in developmental biology, the duration of squint protein expression was photocontrolled in early zebrafish embryos. Squint protein is a nodal-related signal that plays essential roles in organizer development 23 and in the formation of mesodermal and endodermal structures.24 Therefore, ectopic overexpression of squint protein leads to the formation of a secondary axis, but the duplicated axis is incomplete and lacks head structures such as eyes.1 One explanation for these results is that these experimental conditions result in the overexpression of squint protein throughout early development, although squint is normally expressed on the dorsal side only duing the period between the eight-cell stage and the shield stage. I thus examined the influence of the expression period of squint protein on the formation of head structure using mMe2PA-cap. mMe-2PA-capped squint mRNA (5 pg) and 25 pg of mMe-2PA-capped Venus mRNA used as a lineage tracer were coinjected into the cytoplasm of one cell stage embryos (Figure 3a). From the eight-cell stage to 50% epiboly (5.5 hpf), 370 nm light was locally illuminated on the embryos for 2 min every 1 h to induce ectopic expression of squint protein. At the shield stage (6 hpf), 430 nm light was illuminated onto the entire embryo to stop protein expression. A secondary axis with a complete head was observed at 24 hpf in 12% (n = 8/68) of the embryos or 73% (n = 8/11) of the secondary axis embryos (Figure 3b and f). In contrast, I observed a secondary axis

Differences in the translation of mRNA containing either the trans or cis forms of 2PA-cap, pMe-2PA-cap, or mMe-2PA-cap were investigated in zebrafish embryos. Yellow fluorescent protein (Venus) was used to estimate the amount of translated protein. mRNA containing one of the three PC-caps was illuminated with 430 nm light for 2 min to produce the trans form before microinjection into the one cell stage of an embryo. To isomerize PC-cap to the cis form, the embryos were illuminated with 370 nm light for 2 min every 1 h with a 100 W metal halide lamp under a confocal microscope. Translation of mRNA was more efficient with PC-caps in the cis form than in the trans form, as shown in Figure 2. mMe-2PA-cap exhibited the highest photomodulation efficiency. The fluorescence intensity of Venus protein in embryos injected with mMe2PA-capped mRNA and illuminated with 370 nm light was 7.1 times higher than that of nonilluminated embryos, and 76% of protein expression was recovered compared with that of embryos injected with normal-capped mRNA (Figure 2b and c). The smaller amount of translated protein obtained using the trans form of either pMe-2PA-capped or mMe-2PA-capped mRNA compared with 2PA-capped mRNA was likely caused by increased steric hindrance with the active site of eIF4E due to incorporation of methyl groups into the phenyl group. The difference in the amount of translated protein between the cis form of pMe-2PA-cap and mMe-2PA-cap may arise from the difference in the thermal cis-to-trans isomerization rate. On the basis of the above results, we reversibly photoregulated translation in zebrafish embryo using mMe-2PA-cap. The fluorescence intensity of embryos injected with normal-capped Venus mRNA increased at a constant rate (Figure 2d). In contrast, the translation of mMe-2PA-capped Venus mRNA was inhibited until the embryos were illuminated with 370 nm light at 3 h past fertilization (hpf), during which time mMeC

DOI: 10.1021/acschembio.6b00684 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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ACS Chemical Biology

Figure 2. (a) Schematic diagram showing the reversible photoregulation of translation by PC-cap. Translation of mRNA is activated by trans-to-cis photoisomerization of PC-cap upon illumination with 370 nm light. In contrast, cis-to-trans photoisomerization by illumination with 430 nm light inactivates translation. (b) Fluorescence images and (c) average intensity of zebrafish embryos injected with Venus mRNA containing no-cap (n = 20), normal-cap (n = 31), 2PA-cap (n = 33), pMe-2PA-cap (n = 35) or mMe-2PA-cap (n = 35), and 8ST-cap (n = 33) at 24 hpf. The cis forms of the PC-caps were obtained by illumination with 370 nm light for 2 min every 1 h. The cis form of 8ST-cap reported previously20 was obtained by illumination with 410 nm for 2 min soon after microinjection. The average fluorescence intensity of the entire embryo excepting the yolk and standard error was calculated from a result of three independent experiments. Scale bar, 0.5 mm. (d) Time course of Venus protein expression. Black line, embryos injected with normal-capped Venus mRNA (n = 31); blue line, embryos injected with mMe-2PA-capped Venus mRNA and not illuminated with 370 nm light (n = 35); red line, embryos illuminated with 370 nm light for 2 min every 1 h between 3 hpf and 6 hpf and with 430 nm light for 2 min at 7 hpf (n = 22), *P = 0.009 between mMe-2PA-cap (370 nm) and mMe-2PA-cap (370 nm-430 nm); broken red line, embryos illuminated with 370 nm light for 2 min every 1 h between 3 hpf and 9 hpf (n = 22).

without a head structure in 16% (n = 11/70) of the embryos or 92% (n = 11/12) of the secondary axis embryos when the embryos were illuminated with 370 nm light between the eightcell stage and 9 hpf every 1 h and without illumination with 430 nm light (i.e., squint protein was expressed for more than 10 hpf; Figure 3c and f). These results suggest that squint protein leads axis development in the earlier developmental stage and inhibits the formation of head structures when expressed at a later developmental stage. The reason why the induction rate of the secondary axis is low is that the dorsal and ventral side of the embryo cannot be distinguished at the eight-cell stage. The secondary axis was induced only when squint ectopically expresses in the ventral side. The embryos injected no-capped squint mRNA and, illuminated with 370 and 430 nm light, showed a wild-type like phenotype (Figure 3d and f), indicating that 370 and 430 nm light illumination does not affect development of the embryo. When 370 nm light was illuminated to the entire embryo injected mMe-2PA-capped squint mRNA, dorsalized embryos were observed with high frequency. (Figure 3e and f). In summary, PC-caps were synthesized for reversible photoregulation of the translation in vivo. mMe-2PA-cap in the trans form effectively inhibited translation in zebrafish embryos, whereas the cis form yielded protein with a 76% recovery ratio compared with normal-cap, representing a 7.1fold higher efficiency of translation than that of the trans form. In addition, translation activated by illumination with 370 nm

light was rapidly reinactivated by subsequent illumination with 430 nm light. To demonstrate a potential application of this method in developmental biology, I showed photoinduction of the complete secondary axis of zebrafish embryo by regulating the duration of squint protein expression. Moreover, it was found that the formation of head structures was inhibited by prolonged expression of squint. Such findings can only be ascertained using the method for the reversible regulation of translation. Therefore, by using the method described here, we will reveal the importance of when, where, and how long protein expression occurs during specific biological events by spatiotemporally controlling translation in a reversible manner in living cells and organisms.

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METHODS PC-cap Synthesis. The detail of PC-caps synthesis and characterization by NMR and MS is described in the Supporting Information. Photoisomerization. Cis−trans photoisomerization of PCcaps was performed in an aqueous solution containing 75 μM PC-cap at 28.5 °C using a 300 W xenon lamp (MAX-303, Asahi Spectra). Specific wavelengths with a 10 nm peak width at half height were obtained by using an appropriate bandpass filter (LX0370 and MX0430, Asahi Spectra). Light (370 or 430 nm) from the xenon lamp (70% intensity) was illuminated onto a solution of PC-cap for 2 min from a distance of 3 cm D

DOI: 10.1021/acschembio.6b00684 ACS Chem. Biol. XXXX, XXX, XXX−XXX

Letters

ACS Chemical Biology

Figure 3. Photoinduction of the secondary axis in zebrafish embryo. (a) Schematic illustration of the experimental procedure. mMe-2PA-capped squint mRNA (5 pg) and 25 pg of mMe-2PA-capped Venus mRNA used as a lineage tracer were coinjected into the cytoplasm of one cell stage embryos. From the eight-cell stage to 50% epiboly (5.5 hpf), 370 nm light was locally illuminated every 1 h to induce ectopic protein expression. At the shield stage (6 hpf), 430 nm light was illuminated onto the entire embryo to stop protein expression. (b, c) A series of fluorescence and brightfield images showing photoinduction of the secondary axis. Fluorescence shows locally expressed protein and the induced secondary axis in the 24 hpf images. Arrowheads indicate the secondary axis. Scale bar, 100 μm. (b) Embryo illuminated with 370 nm light for 2 min between the eight-cell stage and 5.5 hpf every 1 h and with 430 nm light for 2 min at the shield stage. (c) Embryo illuminated with 370 nm light between the eight-cell stage and 9 hpf every 1 h, but not illuminated with 430 nm light. (d, e) Bright-field images for the embryo illuminated with 370 nm light for 2 min between the eight-cell stage and 5.5 hpf every 1 h onto the entire embryo and with 430 nm light for 2 min at the shield stage. The embryo injected (d) no-capped squint mRNA, (e) mMe-2PA-capped squint mRNA. (f) The frequency of the phenotypes for the embryos.

Capped mRNA Preparation. Venus and squint template DNA containing T7 promoter at 5′ and α-globin 3′-UTR sequence were prepared from pCS2 Venus and pCS2 squint using two-step PCR. PCR amplification was carried out using KOD-plus-DNA polymerase (TOYOBO Co., Ltd.). The in vitro RNA transcription was carried out using the MEGAscriptTM kit (Ambion, Inc.). The reaction solution (20 μL total volume) containing 1 × reaction buffer; 0.5 μg of template DNA; 6 mM each of ATP, CTP, and UTP; 1.2 mM GTP; 4.8 mM PC-cap; and 50 U/μL T7 RNA polymerase was incubated at 37 °C for 4 h. The poly A tailing of synthesized mRNA was carried out using a Poly(A) Tailing Kit (Ambion, Inc.). Purification of the transcribed mRNA from the reaction was performed using the MEGAclearTM kit (Ambion, Inc.). Microinjection and Photoirradiation. Venus or squint mRNA was dissolved in sterile water at a final concentration of 0.05 μg/μL or 0.01 μg/μL, respectively. Venus mRNA or Venus-squint mRNA cocktail was injected into the cytoplasm of a one-cell stage embryo by using FemtoJet (Eppendorf Inc.). Embryos were cultured at 28.5 °C in Ringer’s solution

containing 0.01% kanamycin and penicillin/streptomycin. Photoactivation and inactivation of PC-capped mRNA in zebrafish embryo was conducted as follows. mRNA-injected embryos were transferred to an incubation chamber on the stage of a confocal laser scanning microscope (LSM 710, Carl Zeiss) equipped with a 100 W metal halide lamp (X-Cite 120PC, EXFO) and a bandpass filter (LX0370 or MX0430, Asahi Spectra). Light from the metal halide lamp (25% intensity) was shone on the embryos at 28.5 °C.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00684. Synthesis details of PC-caps, experimental details, and supporting figures (PDF) E

DOI: 10.1021/acschembio.6b00684 ACS Chem. Biol. XXXX, XXX, XXX−XXX

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(16) Yamazoe, S., Liu, Q., McQuade, L. E., Deiters, A., and Chen, J. K. (2014) Sequential Gene Silencing Using Wavelength-Selective Caged Morpholino Oligonucleotides. Angew. Chem., Int. Ed. 53, 10114−10118. (17) Shestopalov, I. A., Sinha, S., and Chen, J. (2007) Lightcontrolled gene silencing in zebrafish embryos. Nat. Chem. Biol. 3, 650−651. (18) Ando, H., Furuta, T., Tsien, R. Y., and Okamoto, H. (2001) Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos. Nat. Genet. 28, 317−325. (19) Gore, A. V., Maegawa, S., Cheong, A., Gilligan, P. C., Weinberg, E. S., and Sampath, K. (2005) The zebrafish dorsal axis is apparent at the four-cell stage. Nature 438, 1030−1035. (20) Ogasawara, S. (2014) Control of Cellular Function by Reversible Photoregulation of Translation. ChemBioChem 15, 2652− 2655. (21) Ogasawara, S., and Maeda, M. (2011) Photoresponsive 5′-cap for the reversible photoregulation of gene expression. Bioorg. Med. Chem. Lett. 21, 5457−5459. (22) Ogasawara, S., Ito, S., Miyasaka, H., and Maeda, M. (2010) Photochromic Nucleobase Photoisomerized by Visible Light. Chem. Lett. 39, 956−957. (23) Feldman, B., Gates, M. A., Egan, E. S., Dougan, S. T., Rennebeck, G., Sirotkin, H., Schier, A. F., and Talbot, W. S. (1998) Zebrafish organizer development and germ-layer formation require nodal-related signals. Nature 395, 181−185. (24) Dougan, S. T., Warga, R. M., Kane, D. A., Schier, A. F., and Talbot, W. S. (2003) The role of the zebrafish nodal-related genes squint and cyclops in patterning of mesendoderm. Development 130, 1837−1851.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Shinzi Ogasawara: 0000-0002-0946-8633 Notes

The author declares no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by PRESTO, JST, and JSPS KAKENHI (Grant Number 16K14733 and 15H00790). We are grateful to Prof. Karuna Sampath (University of Warwick) for providing the pCS2 squint plasmid.

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DOI: 10.1021/acschembio.6b00684 ACS Chem. Biol. XXXX, XXX, XXX−XXX