Abstract
Engineering of translation provides an alternative regulatory layer for controlling transgene expression in addition to transcriptional regulation. Synthetic mRNA switches that modulate translation of a target gene of interest in response to an intracellular protein could be a key regulator to construct a genetic circuit. Insertion of a protein binding RNA sequence in the 5′ UTR of mRNA would allow for the generation of a protein-responsive RNA switch. Here we describe the design principle of the switch and methods for tuning and analyzing its translational activity in mammalian cells.
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References
Isaacs FJ, Dwyer DJ, Collins JJ (2006) RNA synthetic biology. Nat Biotechnol 24:545–554
Wieland M, Fussenegger M (2010) Ligand-dependent regulatory RNA parts for synthetic biology in eukaryotes. Curr Opin Biotechnol 21:760–765
Chang AL, Wolf JJ, Smolke CD (2012) Synthetic RNA switches as a tool for temporal and spatial control over gene expression. Curr Opin Biotechnol 23:679–688
Nandagopal N, Elowitz MB (2011) Synthetic biology: integrated gene circuits. Science 333:1244–1248
Ruder WC, Lu T, Collins JJ (2011) Synthetic biology moving into the clinic. Science 333:1248–1252
Wieland M, Fussenegger M (2012) Engineering molecular circuits using synthetic biology in mammalian cells. Annu Rev Chem Biomol Eng 3:209–234
Ausländer S, Ausländer D, Müller M et al (2012) Programmable single-cell mammalian biocomputers. Nature 487:123–127
Jackson RJ, Hellen CU, Pestova TV (2010) The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol 11:113–127
Sonenberg N, Hinnebusch AG (2009) Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell 136:731–745
Hentze MW, Kühn LC (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. Proc Natl Acad Sci U S A 93:8175–8182
Stripecke R, Oliveira CC, McCarthy JE et al (1994) Proteins binding to 5′ untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells. Mol Cell Biol 14:5898–5909
Paraskeva E, Atzberger A, Hentze MW (1998) A translational repression assay procedure (TRAP) for RNA-protein interactions in vivo. Proc Natl Acad Sci U S A 95:951–956
Nie M, Htun H (2006) Different modes and potencies of translational repression by sequence-specific RNA-protein interaction at the 5′-UTR. Nucleic Acids Res 34:5528–5540
Saito H, Kobayashi T, Hara T et al (2010) Synthetic translational regulation by an L7Ae-kink-turn RNP switch. Nat Chem Biol 6:71–78
Werstuck G, Green MR (1998) Controlling gene expression in living cells through small molecule-RNA interactions. Science 282:296–298
Endo K, Stapleton JA, Hayashi K et al (2013) Quantitative and simultaneous translational control of distinct mammalian mRNAs. Nucleic Acids Res. 41:e135
Imai Y, Matsushima Y, Sugimura T et al (1991) A simple and rapid method for generating a deletion by PCR. Nucleic Acids Res 19:2785
Hansson MD, Rzeznicka K, Rosenbäck M et al (2008) PCR-mediated deletion of plasmid DNA. Anal Biochem 375:373–375
Saito H, Fujita Y, Kashida S et al (2011) Synthetic human cell fate regulation by protein-driven RNA switches. Nat Commun 2:160
Smith AM, Fuchs RT, Grundy FJ et al (2010) Riboswitch RNAs: regulation of gene expression by direct monitoring of a physiological signal. RNA Biol 7:104–110
Eisenstein RS (2000) Iron regulatory proteins and the molecular control of mammalian iron metabolism. Annu Rev Nutr 20:627–662
Stapleton JA, Endo K, Fujita Y et al (2012) Feedback control of protein expression in mammalian cells by tunable synthetic translational inhibition. ACS Synth Biol 1:83–88
Suzuki Y, Ishihara D, Sasaki M et al (2000) Statistical analysis of the 5′ untranslated region of human mRNA using ‘Oligo-Capped’ cDNA libraries. Genomics 64:286–297
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Endo, K., Saito, H. (2014). Engineering Protein-Responsive mRNA Switch in Mammalian Cells. In: Ogawa, A. (eds) Artificial Riboswitches. Methods in Molecular Biology, vol 1111. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-755-6_13
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DOI: https://doi.org/10.1007/978-1-62703-755-6_13
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Publisher Name: Humana Press, Totowa, NJ
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