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http://dx.doi.org/10.14348/molcells.2019.2427

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence  

Lee, Soomin (Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST))
Kim, Jeong-Ah (Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST))
Kim, Hee-Dae (Department of Basic Medical Sciences, University of Arizona College of Medicine-Phoenix)
Chung, Sooyoung (Department of Brain and Cognitive Sciences, Scranton College, Ehwa Womans University)
Kim, Kyungjin (Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST))
Choe, Han Kyoung (Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST))
Publication Information
Molecules and Cells / v.42, no.5, 2019 , pp. 418-425 More about this Journal
Abstract
Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.
Keywords
2A; expression dynamics; internal ribosome entry site; multicistronic elements; real-time fluorescent imaging;
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1 Zhang, R., Lahens, N.F., Ballance, H.I., Hughes, M.E., and Hogenesch, J.B. (2014). A circadian gene expression atlas in mammals: implications for biology and medicine. Proc. Natl. Acad. Sci. U. S. A. 111, 16219-16224.   DOI
2 Arnaud, O., Meyer, S., Vallin, E., Beslon, G., and Gandrillon, O. (2015). Temperature-induced variation in gene expression burst size in metazoan cells. BMC Mol. Biol. 16, 20.   DOI
3 Balvay, L., Soto Rifo, R., Ricci, E.P., Decimo, D., and Ohlmann, T. (2009). Structural and functional diversity of viral IRESes. Biochim. Biophys. Acta 1789, 542-557.   DOI
4 Bouabe, H., Fässler, R., and Heesemann, J. (2008). Improvement of reporter activity by IRES-mediated polycistronic reporter system. Nucleic Acids Res. 36, e28.   DOI
5 Daigle, T.L., Madisen, L., Hage, T.A., Valley, M.T., Knoblich, U., Larsen, R.S., Takeno, M.M., Huang, L., Gu, H., Larsen, R., et al. (2018). A suite of transgenic driver and reporter mouse lines with enhanced brain-cell-type targeting and functionality. Cell 174, 465-480.e22.   DOI
6 Evdokimov, A.G., Pokross, M.E., Egorov, N.S., Zaraisky, A.G., Yampolsky, I.V., Merzlyak, E.M., Shkoporov, A.N., Sander, I., Lukyanov, K.A., and Chudakov, D.M. (2006). Structural basis for the fast maturation of Arthropoda green fluorescent protein. EMBO Rep. 7, 1006-1012.   DOI
7 Fisher, A.C., and DeLisa, M.P. (2008). Laboratory evolution of fast-folding green fluorescent protein using secretory pathway quality control. PLoS One 3, e2351.   DOI
8 Hafner, A., Stewart-Ornstein, J., Purvis, J.E., Forrester, W.C., Bulyk, M.L., and Lahav, G. (2017). p53 pulses lead to distinct patterns of gene expression albeit similar DNA-binding dynamics. Nat. Struct. Mol. Biol. 24, 840-847.   DOI
9 Hellen, C.U., and Sarnow, P. (2001). Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593-1612.   DOI
10 Jang, S.K., Krausslich, H.G., Nicklin, M.J., Duke, G.M., Palmenberg, A.C., and Wimmer, E. (1988). A segment of the 5' nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62, 2636-2643.   DOI
11 Kim, D.Y., Woo, K.C., Lee, K.H., Kim, T.D., and Kim, K.T. (2010). hnRNP Q and PTB modulate the circadian oscillation of mouse Rev-erb alpha via IRESmediated translation. Nucleic Acids Res. 38, 7068-7078.   DOI
12 Kim, J.H., Lee, S.R., Li, L.H., Park, H.J., Park, J.H., Lee, K.Y., Kim, M.K., Shin, B.A., and Choi, S.Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS One 6, e18556.   DOI
13 Komar, A.A., and Hatzoglou, M. (2011). Cellular IRES-mediated translation: the war of ITAFs in pathophysiological states. Cell Cycle 10, 229-240.   DOI
14 Martinez-Salas, E. (1999). Internal ribosome entry site biology and its use in expression vectors. Curr. Opin. Biotechnol. 10, 458-464.   DOI
15 Li, X.L., Li, G.H., Fu, J., Fu, Y.W., Zhang, L., Chen, W., Arakaki, C., Zhang, J.P., Wen, W., Zhao, M., et al. (2018). Highly efficient genome editing via CRISPR-Cas9 in human pluripotent stem cells is achieved by transient BCL-XL overexpression. Nucleic Acids Res. 46, 10195-10215.   DOI
16 Liu, Z., Chen, O., Wall, J.B.J., Zheng, M., Zhou, Y., Wang, L., Ruth Vaseghi, H., Qian, L., and Liu, J. (2017). Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector. Sci. Rep. 7, 2193.   DOI
17 Livet, J., Weissman, T.A., Kang, H., Draft, R.W., Lu, J., Bennis, R.A., Sanes, J.R., and Lichtman, J.W. (2007). Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system. Nature 450, 56-62.   DOI
18 Pelletier, J., and Sonenberg, N. (1988). Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320-325.   DOI
19 Mizuguchi, H., Xu, Z., Ishii-Watabe, A., Uchida, E., and Hayakawa, T. (2000). IRES-dependent second gene expression is significantly lower than capdependent first gene expression in a bicistronic vector. Mol. Ther. 1, 376-382.   DOI
20 Park, K., Jeong, J., and Chung, B.H. (2014). Live imaging of cellular dynamics using a multi-imaging vector in single cells. Chem. Commun. (Camb.) 50, 10734-10736.   DOI
21 R Development Core Team. (2010). R: A Language and Environment for Statistical Computing (Vienna, Austria: R Foundation for Statistical Computing).
22 Ryan, M.D., King, A.M., and Thomas, G.P. (1991). Cleavage of foot-andmouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. J. Gen. Virol. 72 (Pt 11), 2727-2732.   DOI
23 Sage, D., Unser, M., Salmon, P., and Dibner, C. (2010). A software solution for recording circadian oscillator features in time-lapse live cell microscopy. Cell Div. 5, 17.   DOI
24 Sarnow, P. (1989). Translation of glucose-regulated protein 78/immunoglobulin heavy-chain binding protein mRNA is increased in poliovirus-infected cells at a time when cap-dependent translation of cellular mRNAs is inhibited. Proc. Natl. Acad. Sci. U. S. A. 86, 5795-5799.   DOI
25 Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676-682.   DOI
26 Suter, D.M., Molina, N., Gatfield, D., Schneider, K., Schibler, U., and Naef, F. (2011). Mammalian genes are transcribed with widely different bursting kinetics. Science 332, 472-474.   DOI
27 Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567-1572.   DOI
28 Storch, K.F., Lipan, O., Leykin, I., Viswanathan, N., Davis, F.C., Wong, W.H., and Weitz, C.J. (2002). Extensive and divergent circadian gene expression in liver and heart. Nature 417, 78-83.   DOI
29 Sugihara, G., May, R., Ye, H., Hsieh, C.H., Deyle, E., Fogarty, M., and Munch, S. (2012). Detecting causality in complex ecosystems. Science 338, 496-500.   DOI
30 Szymczak, A.L., Workman, C.J., Wang, Y., Vignali, K.M., Dilioglou, S., Vanin, E.F., and Vignali, D.A. (2004). Correction of multi-gene deficiency in vivo using a single 'self-cleaving' 2A peptide-based retroviral vector. Nat. Biotechnol. 22, 589-594.   DOI
31 Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676.   DOI
32 Wickham, H. (2016). ggplot2: Elegant Graphics for Data Analysis (New York: Springer-Verlag New York).
33 Yunger, S., Rosenfeld, L., Garini, Y., and Shav-Tal, Y. (2010). Single-allele analysis of transcription kinetics in living mammalian cells. Nat. Methods 7, 631-633.   DOI
34 Zhang, H., Pu, W., Tian, X., Huang, X., He, L., Liu, Q., Li, Y., Zhang, L., He, L., Liu, K., et al. (2016). Genetic lineage tracing identifies endocardial origin of liver vasculature. Nat. Genet. 48, 537-543.   DOI