Fine Tuning of Translation: A Complex Web of Mechanisms and Its Relevance to Plant Functional Genomics and Biotechnology

Кабардаева, К. В.; Tyurin, A. A.; Pavlenko, O. S.; Gra, O. A.; Deyneko, Igor V.; Kouchoro, F.; Mustafaev, O. N.; Goldenkova-Pavlova, I. V.

doi:10.1134/s1021443719060074

Cited by 9 publications

(8 citation statements)

References 69 publications

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“…Even if the transcription activity of a heterologous gene is maximized, this does not guarantee a high accumulation level of the protein product in the plant. A high level of mRNA of a heterologous gene must be supplemented by an efficient protein synthesis, and this has emerged to be a much more complex problem than was expected [ 7 , 8 ]. The question is how the mRNA translation efficiency of a heterologous gene can be increased.…”

Section: Introductionmentioning

confidence: 99%

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Tyurin

Mustafaev

Suhorukova

et al. 2022

Plants

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A broad and amazingly intricate network of mechanisms underlying the decoding of a plant genome into the proteome forces the researcher to design new strategies to enhance both the accumulation of recombinant proteins and their purification from plants and to improve the available relevant strategies. In this paper, we propose new approaches to optimize a codon composition of target genes (case study of interferon-αA) and to search for regulatory sequences (case study of 5′UTR), and we demonstrated their effectiveness in increasing the synthesis of recombinant proteins in plant systems. In addition, we convincingly show that the approach utilizing stabilization of the protein product according to the N-end rule or a new protein-stabilizing partner (thermostable lichenase) is sufficiently effective and results in a significant increase in the protein yield manufactured in a plant system. Moreover, it is validly demonstrated that thermostable lichenase as a protein-stabilizing partner not only has no negative effect on the target protein activity (interferon-αA) integrated in its sequence, but rather enhances the accumulation of the target protein product in plant cells. In addition, the retention of lichenase enzyme activity and interferon biological activity after the incubation of plant protein lysates at 65°C and precipitation of nontarget proteins with ethanol is applicable to a rapid and inexpensive purification of fusion proteins, thereby confirming the utility of thermostable lichenase as a protein-stabilizing partner for plant systems.

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Section: Introductionmentioning

confidence: 99%

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Tyurin

Mustafaev

Suhorukova

et al. 2022

Plants

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“…The recent advent of new experimental methods, such as RNA-seq and polysome analysis, has allowed for accumulation of a vast body of omics data, making it possible to construct the lists of genes the expression of which at the stages of transcription and translation is induced or regulated by certain factors, in particular, environmental impacts. The insight into the molecular mechanisms underlying the regulation of biological processes in general and gene transcription or mRNA translation in particular, requires the knowledge about the regulatory contexts potentially involved in the intricate mechanisms of gene expression regulation as well as experimental verification of the functional roles of such regulatory contexts in the plant cell [ 1 – 4 ]. A large toolkit of methods is currently available to determine the changes at the level of gene transcription and mRNA translation; their principles, advantages, and disadvantages are disused in several reviews [ 3 , 5 ].…”

Section: Introductionmentioning

confidence: 99%

A high throughput assay of lichenase activity with Congo red dye in plants

et al. 2021

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Background Since the beginning of the use of reporter proteins for expression analysis, a variety of approaches have been developed and proposed; both qualitative and quantitative. The lack of simple methods for direct observation of gene expression in living organisms makes it necessary to continue to propose new methods. In this work, we consider a method for the quantitative analysis of the expression of thermostable lichenase from Clostridium thermocellum used as a sensitive reporter protein. Results In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusions The specific interaction between the dye Congo red and $$\beta$$ β -d-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

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“…The recent advent of new experimental methods, such as RNA-seq and polysome analysis, has allowed for accumulation of a vast body of omics data, making it possible to construct the lists of genes the expression of which at the stages of transcription and translation is induced or regulated by certain factors, in particular, environmental impacts. The insight into the molecular mechanisms underlying the regulation of biological processes in general and gene transcription or mRNA translation in particular, requires the knowledge about the regulatory contexts potentially involved in the intricate mechanisms of gene expression regulation as well as experimental verification of the functional roles of such regulatory contexts in the plant cell [1,2,3,4]. A large toolkit of methods is currently available to determine the changes at the level of gene transcription and mRNA translation; their principles, advantages, and disadvantages are disused in several reviews [3,5].…”

Section: Introductionmentioning

confidence: 99%

A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

Tyurin

Suhorukova

Deineko

et al. 2021

Preprint

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Background : To elucidate the functional role of regulatory sequences and contexts identified and predicted during omics studies in the complex mechanisms of gene expression regulation, experimental verification methods in a plant cell are required. For this, the method of transient expression of reporter genes fused with the tested sequences is widely used. Several reporter systems are available that have shown good performance in the studies including the thermostable lichenase Clostridium thermocellum . However, each reporter system has its own limitations with respect to the quantification of the protein product of a reporter gene and, in particular, for the use of high-throughput approaches. Results: In this study, we report the design a high throughput fluorometric method for quantification of thermostable lichenase C. thermocellum using Congo red and further experimental verification of its relevance and efficiency in assessment of the functional role of regulatory sequences in the plant cell. Conclusion: The specific interaction between the dye Congo red and β -D-glucans formed the background for designing a high-throughput fluorometric assay for quantification of C. thermocellum thermostable lichenase as a reporter protein for plants. This assay (i) makes it possible to precisely measure the amount of reporter protein in a plant sample; (ii) has shown a high sensitivity for quantification of thermostable lichenase; (iii) is more time- and cost-efficient as compared with the Somogyi–Nelson assay; and (iv) is to the least degree dependent on the presence of the tested buffer components as compared with the Somogyi–Nelson assay.

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Fine Tuning of Translation: A Complex Web of Mechanisms and Its Relevance to Plant Functional Genomics and Biotechnology

Cited by 9 publications

References 69 publications

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

Modulation of the Translation Efficiency of Heterologous mRNA and Target Protein Stability in a Plant System: The Case Study of Interferon-αA

A high throughput assay of lichenase activity with Congo red dye in plants

A High Throughput Assay of Lichenase Activity with Congo Red Dye in Plants

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