*4.5. Frameshift Mutations*

The approach of frameshift mutations utilizes concurrent introduction of deletions and insertions at −1 and +1 positions; this procedure changes only the amino acid composition of a peptide sequence coded for in CPuORFs but retains the presence and unchanged length of the overlapping CPuORFs (Figure 7d). This method has been used to analyze 16 recently predicted conserved CPuORFs of *A. thaliana* for assessing a sequence-dependent effect of each CPuORF on expression of the main ORF. A comparative analysis of the reporter protein activity of the variants when the translation is controlled by either native CPuORFs or the CPuORFs with introduced frameshift mutations has identified five novel CPuORFs that repress the expression of the main ORF in a sequence-dependent manner. Moreover, it has been convincingly demonstrated that the C-terminal regions of four of these CPuORF-encoded peptides play a crucial role in repressing the translation of the main ORF [46]. The functionality of three *A. thaliana* CPuORFs in arresting ribosomes during translation was tested in another study. This mechanism of CPuORF action was clarified using toeprinting analysis and

the additional experimental evidence was obtained by constructing the following three types of reporter constructs. (i) With the CPuORF initiation codon removed from each reporter construct of the native CPuORF by replacing AUG with AAG; (ii) with frameshift only mutations, introduced to the CPuORF sequences; and (iii) with both removed initiation codon and frameshift mutations in CPuORF sequences. A comparative testing of all types of reporter constructs has shown that removal of the initiation codon from CPuORFs considerably increases the reporter gene expression; the frameshift mutations in CPuORFs also efficiently increase the reporter gene expression, although to a lower degree as compared with the removal of initiation codon; while the simultaneous presence of frameshift mutations and absence of the initiation codon have almost no effect on the reporter gene expression. These results clearly demonstrate that (i) the peptide sequences are partially responsible for strong repressive effects of these CPuORFs on the main ORF expression; (ii) repression of the main ORF expression (in this case, the ORF of reporter transcript) depends on CPuORF translation; and (iii) these CPuORFs induce ribosomal arrest and thereby considerably inhibit expression of the main ORF [62]. Thus, it is possible not only to insert regulatory sequences that control the reporter gene translation into the constructs carrying this reporter, but also to introduce manifold modifications, which allows their functional role in a key biological process, translation, to be assessed.
