*4.4. Translation from Alternative ORFs*

Translation of the alternative ORFs may be provided by a specific mRNA region, referred to as IRES (internal ribosome entry site). The functionality of an IRES can be experimentally confirmed by constructing bicistronic transcripts (Figure 7e) [40,41]. Note that the use of bicistronic constructs when studying the IRES functionality is determined, in particular, by the need to normalize a relative efficiency of the IRES-guided translation as compared with the cap-dependent translation. For example, functionality of the initially predicted IRES in the mRNA for a tobacco heat shock protein [55] was experimentally confirmed utilizing the ability of coordinated expression of reporter genes within a bicistronic transcript [56]. A structural analysis of the 5'UTR of a maize heat shock protein (Hsp101) mRNA has shown the presence of three stem loops towards the 5' end, which suggested the functioning of the 5'UTR structure as an IRES. This assumption was experimentally confirmed by comparing bicistronic reporter constructs; in particular, it has been shown that the overall hsp101 5'UTR sequence (150 nucleotides) acts as an IRES, since the deletion of 17 nucleotides from the 5' end decreases the translation efficiency of the reporter gene transcript by 87% as compared with the control sequence [57]. A functional analysis of the bicistronic constructs has revealed IRESs in the *A. thaliana* WUS mRNA, coding for the homeodomain transcription factor WUSCHEL (WUS), as well as the role of an additional protein, AtLa1 (an RNA-binding factor). As demonstrated, AtLa1 initiates an IRES-dependent WUS mRNA translation by binding 5'UTR (WUS is responsible for supporting the *A. thaliana* apical meristems under stress conditions) [58].

Alternative ORFs are among the most abundant regulatory elements in mRNAs; they are frequently present in the 5' leader regions of eukaryotic mRNAs (designated uORFs). Such uORFs may negatively modulate the translation efficiency of the downstream main ORF. According to the current estimate, approximately 20% of the *A. thaliana* protein-coding genes contain uORFs in their mRNA 5'UTRs [59]. Initially, such regulatory sequences are either predicted via searching for the conserved peptide uORFs (CPuORFs) [45] or experimentally detected, in particular, by ribosome profiling during, for example, plant cell response to a stress [43,60,61]. The highest interest of researchers is associated with the function of the regulatory sequences, such as CPuORFs, which are able to act in a sequence-dependent manner or cause ribosomal arrest, thereby modulating translation of the main ORF.
