Light-controllable RNA-protein devices for translational regulation of synthetic mRNAs in mammalian cells

Nakanishi, Hiizu; Yoshii, Tatsuyuki; Kawasaki, Shunsuke; Hayashi, Karin; Tsutsui, Keita; Oki, Choji; Tsukiji, Shinya; Saito, Hirohide

doi:10.1016/j.chembiol.2021.01.002

Cited by 23 publications

(32 citation statements)

References 45 publications

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“…Thus, microbial RBPs are primarily used in mRNA-based mammalian synthetic biology. The representative microbial RBPs used to regulate synthetic mRNAs are coat proteins derived from the bacteriophages MS2 (MS2CP) [2][3][4][5][6][10][11][12][13][14][15][16][17][18][19][20][21] and PP7 (PP7CP) [6,14,21], the archaeal ribosomal protein L7Ae [2][3][4]6,7,11,12,[21][22][23][24][25], and the tetracycline-responsive repressor protein (TetR) from Escherichia coli [23,26]. Among these, TetR is unique in its ability to conditionally dissociate from the target RNA motif by doxycycline addition.…”

Section: Binding To Target Mrnasmentioning

confidence: 99%

“…Since caliciviral RNAs lack the 5' cap, caliciviruses use VPg to recruit ribosomes to their RNAs via a VPg-eIF4F interaction [38][39][40]. We showed that the fusion protein of MS2CP and the feline caliciviral VPg, named caliciviral VPg-based translational activator (CaVT), can activate the translation of A-capped mRNAs containing the MS2CP-target motif in their 5' UTRs [5,20] (Figure 2, the 3rd row).…”

Section: Activating Target Mrna Translationmentioning

confidence: 99%

“…Destabilizing domains (also called degrons) that induce rapid degradation of fused proteins are used to regulate the abundance of translational regulator proteins (Figure 2, the 4th row). For example, rapid degradation of a translational repressor protein results in an enhancement of its target mRNA translation [4,6,14,20,23,25]. Conversely, rapid degradation of a translational activator protein diminishes the translation of its target mRNA [20].…”

Section: Destabilizing Proteinsmentioning

confidence: 99%

“…For example, rapid degradation of a translational repressor protein results in an enhancement of its target mRNA translation [4,6,14,20,23,25]. Conversely, rapid degradation of a translational activator protein diminishes the translation of its target mRNA [20]. In many cases, the purpose is to achieve deliberate or cell state-responsive translational regulation by using degrons whose destabilizing activity can be altered by small molecules or endogenous biomolecules.…”

Section: Destabilizing Proteinsmentioning

confidence: 99%

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Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Nakanishi

2021

Life

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Synthetic mRNAs, which are produced by in vitro transcription, have been recently attracting attention because they can express any transgenes without the risk of insertional mutagenesis. Although current synthetic mRNA medicine is not designed for spatiotemporal or cell-selective regulation, many preclinical studies have developed the systems for the translational regulation of synthetic mRNAs. Such translational regulation systems will cope with high efficacy and low adverse effects by producing the appropriate amount of therapeutic proteins, depending on the context. Protein-based regulation is one of the most promising approaches for the translational regulation of synthetic mRNAs. As synthetic mRNAs can encode not only output proteins but also regulator proteins, all components of protein-based regulation systems can be delivered as synthetic mRNAs. In addition, in the protein-based regulation systems, the output protein can be utilized as the input for the subsequent regulation to construct multi-layered gene circuits, which enable complex and sophisticated regulation. In this review, I introduce what types of proteins have been used for translational regulation, how to combine them, and how to design effective gene circuits.

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Section: Binding To Target Mrnasmentioning

confidence: 99%

Section: Activating Target Mrna Translationmentioning

confidence: 99%

Section: Destabilizing Proteinsmentioning

confidence: 99%

Section: Destabilizing Proteinsmentioning

confidence: 99%

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Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Nakanishi

2021

Life

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“…[14] Rapamycin also binds and interferes with the functions of endogenous FKBP and the mammalian target of rapamycin (mTOR), leading to undesired biological effects. To address these issues, several new CID systems have been rationally constructed using two ligand-binding protein tags, including those based on Escherichia coli dihydrofolate reductase (eDHFR) and the FKBP F36V mutant, [15,16] SNAP-tag and (wild-type) FKBP, [17] and eDHFR and HaloTag [16,18–20] pairs. In these systems, chimeric molecules consisting of two small-molecule ligands for each protein tag are used as chemical dimerizers.…”

Section: Introductionmentioning

confidence: 99%

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

Hatano

Suzuki

Nakamura

et al. 2021

Preprint

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Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineered Escherichia coli dihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to the iK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-D-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein–synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonal iK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.

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A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation

2022

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To mimic the levels of spatiotemporal control that exist in nature, tools for chemically induced dimerization (CID) are employed to manipulate protein‐protein interactions. Although linker composition is known to influence speed and efficiency of heterobifunctional compounds, modeling or in vitro experiments are often insufficient to predict optimal linker structure. This can be attributed to the complexity of ternary complex formation and the overlapping factors that impact the effective concentration of probe within the cell, such as efflux and passive permeability. Herein, we synthesize a library of modular chemical tools with varying linker structures and perform quantitative microscopy in live cells to visualize dimerization in real‐time. We use our optimized probe to demonstrate our ability to recruit a protein of interest (POI) to the mitochondria, cell membrane, and nucleus. Finally, we induce and monitor local and global phase separation. We highlight the importance of quantitative approaches to linker optimization for dynamic systems and introduce new, synthetically accessible tools for the rapid control of protein localization.

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Light-controllable RNA-protein devices for translational regulation of synthetic mRNAs in mammalian cells

Cited by 23 publications

References 45 publications

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

Protein-Based Systems for Translational Regulation of Synthetic mRNAs in Mammalian Cells

A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation

A General Strategy for the Design and Evaluation of Heterobifunctional Tools: Applications to Protein Localization and Phase Separation

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