The correlation between transcription levels (abundance of individual mRNAs) and translation (level of corresponding proteins in the proteome) is modest. This confirms that gene expression regulation at the translation stage is crucial. Given the significance of investigations into translationally active mRNA as an indicator of translation effectiveness, researchers require reliable and structured functional sources of information on translation, such as plant translatomic maps that resembling transcription maps. The establishment of a standardized experimental and computational approach is required to generate translatomic maps. In this article, we explore possibilities for researchers to generate translatomic maps for plants. We present a comprehensive overview of experimental methods used to obtain translationally active mRNA fractions, taking into account their advantages and limitations. In addition, we discuss methodological approaches that enable obtaining a complete mRNA pool, sequencing methods for qualitative and quantitative evaluation of various mRNA fractions, as well as bioinformatic methods for multivariate analysis and indicators for assessing mRNA translational activity. This manuscript calls for the scientific community to develop translatomic maps of plant objects as a means to increase the utilization of translatomic data in both fundamental plant biology and practical applications.

Regulation at the level of translation plays an essential role in modulating gene expression. Given the importance of knowledge about translationally active mRNAs as an indicator of translation efficiency, it is crucial for researchers to have at their disposal organised sources of functional information on translation - plant translatomic maps similar to transcription maps. The development of a general experimental and computational methodology for constructing translatome maps is essential to address this challenge. In this manuscript, we discuss what researchers can do to design plant translatome maps, namely, we review: (i) experimental methods to obtain translationally active mRNA fractions, considering their advantages and limitations; (ii) additional methodological approaches to obtain complete pools of translationally active mRNAs; (iii) sequencing methods to qualitatively and quantitatively assess different mRNA fractions; and (iv) multivariate analyses of sequencing results by bioinformatics methods, including metrics for assessing mRNA translational activity.

BACKGROUND:RNA-seq has become a standard technology to quantify mRNA. The measured values usually vary by several orders of magnitude, and while the detection of differences at high values is statistically well grounded, the significance of the differences for rare mRNAs can be weakened by the presence of biological and technical noise.RESULTS:We have developed a method for cleaning RNA-seq data, which improves the detection of differentially expressed genes and specifically genes with low to moderate transcription. Using a data modeling approach, parameters of randomly distributed mRNA counts are identified and reads, most probably originating from technical noise, are removed. We demonstrate that the removal of this random component leads to the significant increase in the number of detected differentially expressed genes, more significant pvalues and no bias towards low-count genes.CONCLUSION:Application of RNAdeNoise to our RNA-seq data on polysome profiling and several published RNA-seq datasets reveals its suitability for different organisms and sequencing technologies such as Illumina and BGI, shows improved detection of differentially expressed genes, and excludes the subjective setting of thresholds for minimal RNA counts. The program, RNA-seq data, resulted gene lists and examples of use are in the supplementary data and at https://github.com/Deyneko/RNAdeNoise .

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.

Additional file 2. R program and examples. The program code of RNAdeNoise in R language, examples of the use.

The polysome profiling method was used to separate mRNAs depending on their loading by ribosomes into polysomal and monosomal fractions. Pools separation of such mRNAs and analysis of transcripts (mRNAs) which are associated with each mRNA pool due to RNA sequencing allowed to get an idea of the translational efficiency of individual mRNAs. Moreover, subsequent in silico analysis make possible searching of regulatory contexts in the 5'-UTR of plant A. thaliana, which may be potentially important for efficient translation of mRNA.

In this study, the polysome profiling method was used for the separation of mRNA depending on their loading by ribosomes into polysomal and monosomal fractions. Separation of pools of such mRNA and analysis of transcripts (mRNA), which are characterized by a constant level of transcription in a wide range of absolute values at all stages of plant ontogenesis and associated with each pool of mRNA due to RNA sequencing, allowed for obtaining an idea about the translational efficiency of individual mRNA. The consequent in silico analysis allowed performing a search for regulatory contexts in the 5'-region of mRNA of Arabidopsis thaliana plants that may be potentially important for efficient mRNA translation. The results of the study revealed that pyrimidine dinucleotides and motifs are characteristic of a 5'-untranslated mRNA region with high translation efficiency, whereas purine dinucleotides and motifs are associated with transcripts with low translational efficiency.

Motivation Polysome profiling is novel, and yet has proved to be an effective approach to detect mRNAs with differential ribosomal load and explore the regulatory mechanisms driving efficient translation. Genes encoding regulatory proteins, having a great influence of the organism, usually reveal moderate to low transcriptional levels, compared, for example, to genes of house-keeping machinery. This complicates the reliable detection of such genes in the presence of technical and/or biological noise.Results In this work we investigate how cleaning of polysome profiling data on Arabidopsis thaliana influences the ability to detect genes with low level of total mRNA, but with a highly differential ribosomal load, i.e. genes translationally active. Suggested data modelling approach to identify a background level of mRNA counts individually for each dataset, shows higher power in detection of low transcribed genes, compared to the use of thresholds for the minimal required mRNA counts or the use of raw data. The significant increase in detected number of regulation–related genes was demonstrated. The described approach is applicable to a wide variety of RNA-seq data. All identified and classified mRNAs with high and low translation status are made available in supplementary material.

We create web database JetGene that allows to estimate the variation of length, nucleotide composition, codon usage frequency and to study nucleotides surrounding of the start codon. JetGene allows user to compare two samples of mRNA.

The paradox of misfit between the levels of mRNAs and their protein products in the eukaryotic cells, including plant cells, encountered by researchers, direct their efforts towards the study into fine mechanisms of translation. Translation is an intricate biological process with numerous players, including mRNAs, tRNAs, ribosomes, and manifold protein factors. Certainly, each of them is important for efficient translation. However, the mRNAs itself contain numerous regulatory elements, such as 5'UTR, the context around the AUG start codon, and codon composition; each element separately or in combination can determine the fate of an individual mRNA in translational process. The previous reviews mainly focus on individual key stages in translation or the aspects of its control. Our goal here is (i) to summarize the recent data on the specific structure–function features of plant mRNAs and their correlation with translational efficiency; (ii) to brief new experimental and theoretical approaches to gaining the insight into the complex network of translation; and (iii) to assess the relevance of this knowledge to both the plant functional genomics and biotechnological application.

The control of translation in the course of gene expression regulation plays a crucial role in plants’ cellular events and, particularly, in responses to environmental factors. The paradox of the great variance between levels of mRNAs and their protein products in eukaryotic cells, including plants, requires thorough investigation of the regulatory mechanisms of translation. A wide and amazingly complex network of mechanisms decoding the plant genome into proteome challenges researchers to design new methods for genome-wide analysis of translational control, develop computational algorithms detecting regulatory mRNA contexts, and to establish rules underlying differential translation. The aims of this review are to (i) describe the experimental approaches for investigation of differential translation in plants on a genome-wide scale; (ii) summarize the current data on computational algorithms for detection of specific structure–function features and key determinants in plant mRNAs and their correlation with translation efficiency; (iii) highlight the methods for experimental verification of existed and theoretically predicted features within plant mRNAs important for their differential translation; and finally (iv) to discuss the perspectives of discovering the specific structural features of plant mRNA that mediate differential translation control by the combination of computational and experimental approaches.

Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.

The main specific features of β-1,3-1,4-glucanases (or lichenases, EC 3.2.1.73), the enzymes that in a strictly specific manner hydrolyze β-glucans of many cereal species and lichens containing β-1,3 and β-1,4 bonds, are reviewed as well as the current strategies used for their protein design, which have been successfully applied to make lichenases more attractive and promising for biocatalytic conversion of biomass, in particular, in the areas of their biotechnological application, such as brewing industry, animal feed manufacture, and biofuel production, which will in future allow these technologies to be economically and ecologically beneficial.

Аннотация.Диссонанс между уровнями мРНК и белка заботит исследователей с самого начала генно-инженерной эпохи.Однако ввиду ряда причин основной акцент в подобных исследованиях до последнего десятилетия

A recombinant DNA in which the interferon αA (IFN–αA) gene sequence is integrated into a loop region of the gene coding thermostable lichenase was constructed. This approach of insertion fusion with thermostable lichenase is advantageous in terms of increasing the solubility, stability, and production of the fusion partner in soluble form in general and in the periplasm of bacterial cells in particular. Thus, the insertion of IFN–αA into the loop (53 a.a.) of thermostable lichenase from Clostridium thermocellum resulted in effective expression of the soluble form of the recombinant protein in the periplasm of Escherichia coli without any compromise in biological activity of IFN–αA, while the thermostable lichenase retained its ability for functional folding without dramatic loss of its basic activity and thermostability.