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

Life ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1192
Author(s):  
Hideyuki Nakanishi
Keyword(s):  
Adverse Effects ◽  
Mammalian Cells ◽  
Translational Regulation ◽  
Insertional Mutagenesis ◽  
In Vitro Transcription ◽  
Preclinical Studies ◽  
High Efficacy ◽  
Gene Circuits ◽  
Synthetic Mrna

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.

scholarly journals Construction of a synthetic messenger RNA encoding a membrane protein.

1983 ◽  
Vol 96 (5) ◽  
pp. 1464-1469 ◽  
Author(s):  
J L Rubenstein ◽  
T G Chappell
Keyword(s):  
G Protein ◽  
Messenger Rna ◽  
In Vitro Transcription ◽  
Membrane Insertion ◽  
Wheat Germ Extract ◽  
E Coli ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Synthetic Mrna

We have synthesized microgram quantities of a functional eucaryotic mRNA by in vitro transcription. For this purpose, we constructed a plasmid in which the Escherichia coli lactose promoter was 5' to the vesicular stomatitis virus (VSV) G protein gene (Rose, J. K., and C. J. Gallione, 1981, J. Virol., 39:519-528). This DNA served as the template in an in vitro transcription reaction utilizing E. coli RNA polymerase. The RNA product was capped using the vaccinia guanylyltransferase. A typical preparation of the synthetic G mRNA was equivalent to the amount of G mRNA that can be isolated from approximately 10(8) VSV-infected cells. This synthetic mRNA was translated by a wheat germ extract in the presence of microsomes, producing a polypeptide that was indistinguishable from G protein in its size, antigenicity, degree of glycosylation, and its membrane insertion. This technique should aid in identifying features needed by proteins for insertion into membranes.

scholarly journals The focal-adhesion vasodilator-stimulated phosphoprotein (VASP) binds to the proline-rich domain in vinculin

1996 ◽  
Vol 318 (3) ◽  
pp. 753-757 ◽  
Author(s):  
Nicholas P. J. BRINDLE ◽  
Mark R. HOLT ◽  
Joanna E DAVIES ◽  
Caroline J PRICE ◽  
David R. CRITCHLEY
Keyword(s):  
Focal Adhesion ◽  
Mammalian Cells ◽  
Focal Adhesions ◽  
In Vitro Transcription ◽  
Translation System ◽  
Rich Region ◽  
Rich Domain ◽  
Focal Adhesion Protein ◽  
Proline Rich Region

In mammalian cells vasodilator-stimulated phosphoprotein (VASP) is localized to focal adhesions and areas of dynamic membrane activity where it is thought to have a role in actin-filament assembly. The proteins responsible for recruiting VASP to these sites within the cell are not known. The bacterial protein ActA binds VASP via a proline-rich motif that is very similar to a sequence in the proline-rich region of the focal-adhesion protein vinculin. We have examined the ability of VASP, synthesized using an in vitro transcription/translation system, to bind to a series of vinculin peptides expressed as glutathione S-transferase fusion proteins, and have shown that it binds specifically to the proline-rich region in vinculin. Using immobilized peptides corresponding to the two proline-rich motifs within this domain, the VASP-binding site was localized to proline-rich motif-1 (residues 839–850). Binding to this motif was not affected by the phosphorylation state of VASP. The C-terminal region of VASP, which is known to be important in targeting VASP to focal adhesions, was shown to be required for binding. These results identify vinculin as a VASP-binding protein likely to be important in recruiting VASP to focal adhesions and the cell membrane.

scholarly journals The regulatory enzymes and protein substrates for the lysine β-hydroxybutyrylation pathway

2021 ◽  
Vol 7 (9) ◽  
pp. eabe2771 ◽  
Author(s):  
He Huang ◽  
Di Zhang ◽  
Yejing Weng ◽  
Kyle Delaney ◽  
Zhanyun Tang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Functional Study ◽  
Histone Deacetylase 1 ◽  
Major Mechanism ◽  
Cellular Processes ◽  
Regulatory Enzymes ◽  
Substrate Proteins

Metabolism-mediated epigenetic changes represent an adapted mechanism for cellular signaling, in which lysine acetylation and methylation have been the historical focus of interest. We recently discovered a β-hydroxybutyrate–mediated epigenetic pathway that couples metabolism to gene expression. However, its regulatory enzymes and substrate proteins remain unknown, hindering its functional study. Here, we report that the acyltransferase p300 can catalyze the enzymatic addition of β-hydroxybutyrate to lysine (Kbhb), while histone deacetylase 1 (HDAC1) and HDAC2 enzymatically remove Kbhb. We demonstrate that p300-dependent histone Kbhb can directly mediate in vitro transcription. Moreover, a comprehensive analysis of Kbhb substrates in mammalian cells has identified 3248 Kbhb sites on 1397 substrate proteins. The dependence of histone Kbhb on p300 argues that enzyme-catalyzed acylation is the major mechanism for nuclear Kbhb. Our study thus reveals key regulatory elements for the Kbhb pathway, laying a foundation for studying its roles in diverse cellular processes.

scholarly journals Ferric Enterochelin Transport in Yersinia enterocolitica: Molecular and Evolutionary Aspects

1999 ◽  
Vol 181 (20) ◽  
pp. 6387-6395 ◽  
Author(s):  
S. Schubert ◽  
D. Fischer ◽  
J. Heesemann
Keyword(s):  
Gene Cluster ◽  
Yersinia Enterocolitica ◽  
Insertional Mutagenesis ◽  
Yersinia Pseudotuberculosis ◽  
Functional Characterization ◽  
In Vitro Transcription ◽  
E Coli ◽  
The Family

ABSTRACT Yersinia enterocolitica is well equipped for siderophore piracy, encompassing the utilization of siderophores such as ferrioxamine, ferrichrome, and ferrienterochelin. In this study, we report on the molecular and functional characterization of theYersinia fep-fes gene cluster orthologous to theEscherichia coli ferrienterochelin transport genes (fepA, fepDGC, and fepB) and the esterase gene fes. In vitro transcription-translation analysis identified polypeptides of 30 and 35 kDa encoded byfepC and fes, respectively. A frameshift mutation within the fepA gene led to expression of a truncated polypeptide of 40 kDa. The fepD,fepG, and fes genes of Y. enterocolitica were shown to complement corresponding E. coli mutants. Insertional mutagenesis of fepD orfes genes abrogates enterochelin-supported growth ofY. enterocolitica on iron-chelated media. In contrast toE. coli, the fep-fes gene cluster inY. enterocolitica consists solely of genes required for uptake and utilization of enterochelin (fep) and not of enterochelin synthesis genes such as entF. By Southern hybridization, fepDGC and fes sequences could be detected in Y. enterocolitica biotypes IB, IA, and II but not in biotype IV strains, Yersinia pestis, andYersinia pseudotuberculosis strains. According to sequence alignment data and the coherent structure of the Yersinia fep-fes gene cluster, we suggest early genetic divergence of ferrienterochelin uptake determinants among species of the familyEnterobacteriaceae.

185 CELLULAR VIABILITY AND EXPRESSION OF GREEN FLUORESCENT PROTEIN IN BOVINE FETAL FIBROBLASTS FOLLOWING TRANSFECTION OF SYNTHETIC mRNA INCLUDING MODIFIED BASES

2014 ◽  
Vol 26 (1) ◽  
pp. 207
Author(s):  
T. L. Adams ◽  
S. E. Farmer ◽  
J. A. Sarmiento-Guzmán ◽  
K. R. Bondioli
Keyword(s):  
Fluorescent Protein ◽  
In Vitro Transcription ◽  
Cellular Reprogramming ◽  
Cellular Viability ◽  
Treatment Groups ◽  
Fetal Fibroblasts ◽  
Green Fluorescent ◽  
Synthetic Mrna ◽  
Number Of Cells

Synthetic RNA transfection has been an invaluable tool in understanding the mammalian genome because of its ability to deliver exogenous protein without mutagenic effects caused by double-stranded DNA. A common problem associated with the introduction of exogenous mRNA into mammalian cells is the stimulated interferon response. This innate immune response can be avoided with the addition of modified bases during the in vitro transcription process of synthetically derived mRNA. The bases cytidine triphosphate (CTP) and uridine triphosphate (UTP) are replaced with 5-methylcytidine-5′-triphosphate (5-Methyl-CTP) and pseudouridine-5′-triphosphate (Pseudo-UTP) during in vitro transcription. Cellular reprogramming is achieved by the delivery of this mRNA into the cytoplasm. Previous cellular reprogramming experiments lacking modified bases resulted in increased toxicity and a decrease in cellular viability, which lead to the incorporation of modified bases. In the first experiment, bovine fetal fibroblasts were transfected with modified synthetic mRNA encoding green fluorescent protein (GFP) to evaluate the effects on cellular viability and fluorescence. The cellular viability was measured by counting a final number of cells after seeding a constant number of cells in all treatment groups. The control group consisted of bovine fetal fibroblasts cultured in normal growth medium. A no-RNA (NR) group was held under the same conditions with the addition of the transfection reagent, Lipofectamine (Invitrogen, Carlsbad, CA, USA), to account for toxicity resulting from the transfection reagent alone. The cells were transfected every other day for 12 days and were evaluated on days 3, 6, 9, and 12 for viability and fluorescence by flow cytometry. There was no difference in viability of all cells treated with synthetic mRNA encoding GFP when compared to controls (P = 0.9). There was a significant difference in fluorescence on all time points when compared to controls (Day 3, P = 0.004; Day 6, P = 0.004; Day 9, P = 0.007; Day 12, P = 0.04). The second experiment consisted of bovine fetal fibroblasts transfected with modified synthetic mRNA encoding pluripotency factors. The controls were identical to the previous experiment, but treatment groups were transfected with modified synthetic mRNA encoding either three factors (3F: OCT4, SOX2, KLF4) or four factors (4F: OCT4, SOX2, KLF4, c-MYC). The treated cells were transfected every other day and evaluated on Day 24 for cellular viability. There was no difference in cellular viability in all treatment groups when compared to controls (P = 0.2). The introduction of synthetic mRNA containing modified bases maintains cellular viability when compared to controls. The decreased immune response by the inclusion of modified bases may be advantageous in a variety of applications from the introduction of transcription activator-like effector nuclease (TALEN) or zinc finger nucleases for genomic editing to increased efficiency of the development of induced pluripotent stem cells.

scholarly journals Noise-reducing optogenetic negative-feedback gene circuits in human cells

2019 ◽  
Vol 47 (14) ◽  
pp. 7703-7714 ◽  
Author(s):  
Michael Tyler Guinn ◽  
Gábor Balázsi
Keyword(s):  
Gene Expression ◽  
Noise Reduction ◽  
Negative Feedback ◽  
Mammalian Cells ◽  
Cellular Proliferation ◽  
Protein Domain ◽  
Gene Circuits ◽  
Expression Control ◽  
Gene Expression Control

Abstract Gene autorepression is widely present in nature and is also employed in synthetic biology, partly to reduce gene expression noise in cells. Optogenetic systems have recently been developed for controlling gene expression levels in mammalian cells, but most have utilized activator-based proteins, neglecting negative feedback except for in silico control. Here, we engineer optogenetic gene circuits into mammalian cells to achieve noise-reduction for precise gene expression control by genetic, in vitro negative feedback. We build a toolset of these noise-reducing Light-Inducible Tuner (LITer) gene circuits using the TetR repressor fused with a Tet-inhibiting peptide (TIP) or a degradation tag through the light-sensitive LOV2 protein domain. These LITers provide a range of nearly 4-fold gene expression control and up to 5-fold noise reduction from existing optogenetic systems. Moreover, we use the LITer gene circuit architecture to control gene expression of the cancer oncogene KRAS(G12V) and study its downstream effects through phospho-ERK levels and cellular proliferation. Overall, these novel LITer optogenetic platforms should enable precise spatiotemporal perturbations for studying multicellular phenotypes in developmental biology, oncology and other biomedical fields of research.

scholarly journals A novel role for kynurenine 3-monooxygenase in mitochondrial dynamics

PLoS Genetics ◽  
2020 ◽  
Vol 16 (11) ◽  
pp. e1009129
Author(s):  
Daniel C. Maddison ◽  
Mónica Alfonso-Núñez ◽  
Aisha M. Swaih ◽  
Carlo Breda ◽  
Susanna Campesan ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Translational Regulation ◽  
Metabolic Flux ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Kynurenine Pathway ◽  
Loss Of Function ◽  
Disease Associated Genes

The enzyme kynurenine 3-monooxygenase (KMO) operates at a critical branch-point in the kynurenine pathway (KP), the major route of tryptophan metabolism. As the KP has been implicated in the pathogenesis of several human diseases, KMO and other enzymes that control metabolic flux through the pathway are potential therapeutic targets for these disorders. While KMO is localized to the outer mitochondrial membrane in eukaryotic organisms, no mitochondrial role for KMO has been described. In this study, KMO deficient Drosophila melanogaster were investigated for mitochondrial phenotypes in vitro and in vivo. We find that a loss of function allele or RNAi knockdown of the Drosophila KMO ortholog (cinnabar) causes a range of morphological and functional alterations to mitochondria, which are independent of changes to levels of KP metabolites. Notably, cinnabar genetically interacts with the Parkinson’s disease associated genes Pink1 and parkin, as well as the mitochondrial fission gene Drp1, implicating KMO in mitochondrial dynamics and mitophagy, mechanisms which govern the maintenance of a healthy mitochondrial network. Overexpression of human KMO in mammalian cells finds that KMO plays a role in the post-translational regulation of DRP1. These findings reveal a novel mitochondrial role for KMO, independent from its enzymatic role in the kynurenine pathway.

298 STIMULATION OF ENDOGENOUS OCT4 IN BOVINE FETAL FIBROBLASTS WITH SYNTHETIC mRNA TRANSFECTION

2013 ◽  
Vol 25 (1) ◽  
pp. 296
Author(s):  
T. L. Adams ◽  
S. E. Farmer ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Cell Viability ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
In Vitro Transcription ◽  
Cell Number ◽  
Rt Pcr ◽  
Time Points ◽  
Fetal Fibroblasts ◽  
Synthetic Mrna

Induced pluripotent cells have been produced with viral vectors and other transgenic approaches that modify the genome of the host cell. Transfection with mRNA encoding transcription factors eliminates genomic modification and has been successful with human cells. The objective of this study was to deliver synthetic mRNA to bovine fetal fibroblasts in a manner consistent with induced pluripotency. Plasmids encoding green fluorescent protein (GFP) and human OCT4 were used as templates for in vitro transcription of mRNA (ivtRNA) and used to transfect bovine fetal fibroblasts. Expression of GFP was measured by flow cytometry and cell viability by seeding a constant cell number and determining final cell count. Differences between treatments were tested by ANOVA at P ≤ 0.05. In the first experiment, ivtRNA concentrations of 140, 280, and 560 ng/well of a 24-well plate were used and cell number and fluorescence determined after 24 h. Transfected treatments had significantly fewer cells and greater fluorescence than controls. Cells transfected with 280 ng/well ivtRNA had the highest fluorescence. Cells were then transfected with 280 ng/well of ivtRNA and total cells and fluorescence determined after 12, 24, 36, 48, and 60 h. Cell number was significantly lower than controls for all time points but similar between time points. Fluorescence was greater than controls for all time points and peaked at 24 to 36 h. There was no difference in fluorescence between 24 and 36 h, but these time points were significantly greater than all other time points. From these data, we determined that sustained expression could be maintained with transfection every other day. Cells were transfected every other day with ivtRNA encoding GFP and fluorescence measured every 3 days. Fluorescence was greater than controls for all time points but did not increase with repeated transfection. After 6 transfections, there were no viable cells left. In the final experiment, cells were transfected every other day for 12 days with 140, 280, or 560 ng/well ivtRNA encoding human Oct4. After 12 days, RNA was isolated and bovine OCT4 transcripts measured by RT-PCR. Primers and PCR conditions were designed so that bovine OCT4 transcripts were amplified but human OCT4 ivtRNA was not. The bovine poly-adenylate polymerase (PAP) transcript was also amplified. After gel electrophoresis of RT-PCR products, band intensities were determined and a ratio of OCT4/PAP calculated. This ratio was 0.7 for controls and 1.2 for the 140 ng/well ivtRNA treatment. No bovine OCT4 expression was detected in the higher ivtRNA treatments because of poor cell viability. From these experiments, we conclude that ivtRNA can be transfected into and expressed by bovine fetal fibroblasts. Repeated transfections with ivtRNA encoding human OCT4 induced expression of the endogenous bovine OCT4 but cell viability was reduced. Further experiments utilising ivtRNA with modified bases are planned to reduce the cell toxicity of ivtRNA.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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