*4.1. Direct Measurments of Individual Transcripts*

Several tools for determining the changes of individual transcripts at the level of translation are available, including (i) the systems for in vitro translation based on measuring the incorporation of labeled amino acids, such as FUNCAT (fluorescent noncanonical amino acid tagging), SILAC (stable isotope labeling by amino acids in cell culture), BONCAT (bioorthogonal noncanonical amino acid tagging), QuaNCAT (quantitative noncanonical amino acid tagging), and PUNCH-P (puromycin-associated nascent chain proteomics), or cell-free protein expression systems, such as the wheat-germ extract, which contain all factors necessary for translation of the target transcript; (ii) toeprinting, or the primer extension inhibition assay, utilizing reverse transcription to study the interaction of ribosomes with the target transcript; (iii) enzyme immunoassays, in particular, Western blot hybridization; and (iv) mass spectrometry-based methods for identifying the changes in the proteome or their combinations with in vitro translation methods, for example, PUNCH-P. The review by Mazzoni-Putman et al. [4] describes the principles, advantages, and limitations of these methods in detail.

However, these research methods are in most cases suitable for assessing the general changes in translation, require considerable time, amount of reagents, and specialized equipment; correspondingly, most research works now use the strategy of reporter systems for studying the structure–function characteristics of the target sequences. The strategy of reporter genes considerably enhances such research since it is much easier to record the protein product of a reporter gene as compared with a studied gene. It should be also emphasized that the reporter genes code for the proteins that display either unique specific features or unique enzyme activities, allowing their products to be easily isolated from the totality of intracellular and extracellular proteins. Thanks to these advantages of reporter systems over the other methods for studying the regulation of gene expression, they have been widely used for experimental verification of the regions and nucleotide contexts in differentially-translated transcripts. For studies of this type, expression cassettes are constructed that carry the reporter gene sequence with the expression controlled by a particular regulatory region or sequence selected by researcher (Figure 7). Researchers have at their disposal several reporter systems that have proved their efficiency in the studies of potential regulatory sequences or the nucleotide contexts that modulate translation in plant systems, in particular, β-glucuronidase (GUS); different variants of fluorescent proteins (for example, GFP and RFP); luciferases (Renilla luciferase, RLuc, and firefly luciferase, FLuc); and thermostable lichenase (LicBM) [51,52]. Commercial substrates and kits as well as the quantification methods for assessing the corresponding protein products are available for these reporter systems. The main approaches that have been applied in the studies of various regulatory regions or nucleotide contexts within transcripts with the use of reporter systems are illustrated below.

When experimentally confirming the role of the full-sized 5'UTRs in transcripts, these sequences are cloned upstream of the 5' region of a reporter gene (Figure 7). Quantitative estimate of the reporter gene protein product when using different target 5'UTRs versus the known translational enhancers of various origins makes it possible to assess their contribution to the translation efficiency [51]. For example, *A. thaliana* mRNAs that are stably translated under any growth and environmental conditions have been found by polysome profiling. Testing of the translation capability of mRNA 5'UTRs of candidate genes using the reporter gene strategy has convincingly demonstrated that the 5'UTR of 47 cold-regulated genes are an efficient translational enhancers, which enables a stable high level translation under any conditions of plant growth. This suggests the utility of this method for plant biotechnology, for example, when engineering plants producing biologically active substances or the plants resistant to some stress factors, including the schemes that involve genome editing technologies [9].

Recombinant 5'UTR sequences are designed for identification of the cis-regulatory elements in these mRNA regions, first and foremost, the motifs or specific nucleotide contexts potentially able to influence differential translation, using for this purpose a (i) combinatorial approach, (ii) site-specific mutagenesis, (iii) translation assessment of the second (3'-terminal) ORF of a bicistronic transcript, (iv) deletion analysis, (v) frameshift mutations, or a combination of these methods (Figure 7).

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