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.

A bireporter vector for the study of translational cis-regulatory elements has been created and tested.

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.

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.

The efficiency of transient gene expression in plants credibly demonstrated characteristics of gene functions in numerous studies. Two key strategies of transient expression became favorites among researchers: protoplast transfection and agroinfiltration. Each of them, alongside the advantages, has its own constraints. In this work, an easy, rapid, and reliable system for characterization of the signal sequences and determinations of target protein localization in a plant cell is proposed and tested. This system—called the AgI–PrI—implies production of protoplasts from plant tissues after agroinfiltration. Reliability of the proposed system for transient gene expression has been proved using characterized signal sequences in Nicotiana benthamiana cells. The corresponding protocol is less expensive and depends to a lesser degree on the professional skills in the area of protoplast isolation and transfection; furthermore, it may be applicable to other plant species with either available efficient methods of agroinfiltration and protoplast isolation or with the potential for one of the protocols to be supplemented. Thus, the AgI–PrI technique makes it possible to combine the advantages of two widely used methods for the transient gene expression in plants—agroinfiltration and protoplast isolation and transfection—and concurrently avoids their critical points.

The contribution of nucleotide composition of mRNA 5'-region to the efficiency of expression at transcriptional and translational levels was studied in transgenic tobacco plants ( Nicotiana tabacum L., cultivar Petit Havana) using a thermostable lichenase reporter gene. Synthetic sequence that contains CG-rich motifs, typical for 5'-region of plant genes, identified in silico , was constructed. Transgenic plant lines of N. tabacum were obtained; they contain thermostable lichenase reporter gene that is under control of the constitutive 35S RNA CaMV promoter and additional regulatory element: synthetic CG-rich sequence, which functions as the 5'-UTR (untranslated region) of the reporter gene mRNA or as 5'-region of the hybrid gene coding sequence, wherein the synthetic sequence is fused with the sequence of the reporter gene in its reading frame. Results of the comparative analysis of mRNA and protein levels in the obtained lines of transgenic plants showed that the synthetic CG-rich sequence significantly increases the level of transcription of the reporter gene and appear to have no negative effect on the efficiency of reporter mRNA translation, which may be due to the peculiarities of its nucleotide composition and structure, namely, due to the presence of motifs that are specific for 5'-regions of plant genes, as well as due to the properties of the secondary structure— the absence of hairpin structures with a high energy of formation. It was experimentally confirmed for the first time that the 5'-region of the genes with a high content of CpG dinucleotides can help to increase the transcription level of genes in plants.

The results of bioinformatics analysis allowed to reveal the following consistent pattern: the average length of the 5'-untranslated region (5'-UTRs) for most A. thaliana's genes with high expression levels range from 80 to 120 bp, with an average GC content of 36,5%. Based on alignment results was defined motive in 5'-UTRs, as a new regulatory element, which could potentially provide highly efficient expression and synthesis of the target product in plants. This sequence has a length of 87 bp and GC content of 35.6%. It was demonstrated that consensus sequence of 5'-UTRs increased accumulation of the bi-reporter protein more than 25%, by that acting as a potential positive regulatory element on translational efficiency.