||Analytical study on the peptide sequence-dependent regulatory upstream open reading frame of a tomato homologue of the Arabidopsis ANAC096 gene [an abstract of entire text]
NOH, ABDUL LATIF BIN
Many eukaryotic mRNAs contain one or more upstream open reading frames (uORFs)in their 5′ untranslated regions (5′-UTRs). Some uORFs encode regulatory peptides thatrepress translation of the main ORF. To comprehensively search for uORFs encodingregulatory peptides, uORFs with evolutionarily conserved amino acid sequences,referred to as conserved peptide uORFs (CPuORFs), have been identified usingbioinfomatic approaches.The Arabidopsis thaliana ANAC096 gene is one of the CPuORF-containing genes;however, the ANAC096 CPuORF exerts only little peptide sequence-dependent effecton expression of the main ORF. This project focused on the effect of the CPuORFsequence of a tomato ANAC096 homologue, LOC101264451, on expression of the mainORF, because it has a more highly conserved amino acid sequence than the ANAC096CPuORF.In this study, to address the importance of the CPuORF amino acid sequence for theregulatory function, mutational analyses on the LOC101264451 CPuORF sequencewere conducted, and the effects on main ORF expression were examined using transientexpression assay with protoplasts prepared from tobacco BY-2 cultured cells. Alterationof the CPuORF amino acid sequence by a frameshift (fs) mutation conferred more thantwo-fold increase in main ORF expression compared with the wild-type (WT). Theeffect of the fs mutation was abolished in the absence of the CPuORF start codon. Thisresult indicates that translation of the CPuORF is required for the fs mutation to exert itseffect, and suggests that the effect of the fs mutation is caused by the amino acidsequence alteration of the CPuORF rather than by the nucleotide sequence change.Furthermore, to determine the critical amino acid residues of the LOC101264451CPuORF peptide responsible for the regulation, alanine scanning analysis wasperformed. Most of the Ala substitutions introduced into the conserved region showed asignificant increase in the reporter activity compared with the WT. By contrast,synonymous codon changes introduced into the similar region showed only a slightincrease in the reporter activity. These observations suggest that the peptide encoded bythe LOC101264451 CPuORF is involved in the repression of main ORF expression.The ANAC096 gene encodes a NAC (NAM, ATAF1,2 and CUC2)domain-containing transcription factor. The expression of this gene is induced at themRNA level in response to dehydration and osmotic stress in A. thaliana. Since theLOC101264451 main ORF is orthologous to ANAC096, the expression of the mainORF may be induced at the post-transcriptional level in response to similar stresses, andthe CPuORF may be involved in the regulation. To address the effect of theLOC101264451 CPuORF on main ORF expression under stress conditions, the reporterplasmid carrying the CPuORF upstream of a luciferase gene was transfected into BY-2protoplasts, and the protoplasts were incubated with several different concentration ofmannitol. The inhibitory effect of the CPuORF on main ORF expression was dependenton mannitol concentration. This result suggests that the LOC101264451 CPuORF isinvolved in post-transcriptional regulation in response to osmotic stress.
Hokkaido University（北海道大学）. 博士(農学)