S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. 5´-leader sequence of carnation SAMDC (CSDC9) mRNA contains two overlapping tiny (2 AAs) and small uORFs (54 AAs). To explore the role of tiny and small uO...
S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. 5´-leader sequence of carnation SAMDC (CSDC9) mRNA contains two overlapping tiny (2 AAs) and small uORFs (54 AAs). To explore the role of tiny and small uORFs of SAMDC gene in controlling transcription and/or translation of downstream ORF, chimeric genes were constructed containing the point-mutated forms of the uORF sequences fused to the GUS reporter gene. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF peptide. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was showed more functional importance as a translational inhibitor, compared with tiny uORF sequence. Importantly, the small uORF sequence induced a real peptide of 5.7kDa, which was provided the presence of SAMDC small uORF peptide band using an in vitro transcription/translation system. Therefore, the peptide products specified by these uORFs might cause ribosomes that are translating them to stall in a sequence-dependent manner. In addition in cis repressive effect, uORF inhibited the translation of a downstream ORF in trans. The expression levels of transcripts containing uORF were markedly lower than those not containing uORF. Moreover, the half-life for degradation of the uORF-containing transcripts was also shorter. These results suggest that uORFs are sequence elements that down-regulate RNA transcripts via RNA decay mechanisms. Also, we showed that degradation rate of GUS and SAMDC protein after treatment with cycloheximide and specific proteasome inhibitor MG115 was accelerated in the presence of small uORF peptide. However, these effects of SAMDC small uORF peptide did not occurred on endogenous activity of ADC, which was other enzyme for polyamine biosynthesis and contained its own ADC uORF sequence in 5’UTR. Therefore, SAMDC small uORF has a sequence-specific function of transcriptional and/or translational inhibitor for its own SAMDC protein not only in cis but also in trans. Also, we showed that the uORF peptide specifically was phosphorylated by unknown kinase in cell-free system. Putative phosphorylation sites for protein kinase C, cAMP and cGMP dependent kinase and casein kinase Ⅱ in uORF peptide might be important for inhibitory effect of uORF. To investigate the function of the phosphorylated uORF peptide, chimeric genes were constructed containing the several phosphorylation site-mutated forms of the uORF sequences fused to the GUS reporter gene. The translational level was completely retained in phosphorylated protein of Ser17 (P17), which is a putative site for protein kinase C. Therefore, it is suggested that protein kinase C might have an important role for acting a translational inhibitor of downstream ORF. Also, RNA and protein degradation rate of downstream GUS were significantly delayed in transgenic plant without unphosphorylated small uORF than the other transgenic plants with phosphorylated uORF. These results implied that phosphorylated small uORF peptide might effectively act as a functional regulator in RNA and protein degradation. This is the first report to provide evidence that phosphorylation of small uORF may repress the translation of downstream ORF in sequence-dependent manner in plant mRNAs. Therefore it contributes to a broader understanding of the role plated by uORFs in the regulation of gene expression.
S-Adenosylmethionine decarboxylase (SAMDC; EC 4.1.4.50), a key enzyme for polyamines biosynthesis, was tightly regulated for homeostatic levels. 5´-leader sequence of carnation SAMDC (CSDC9) mRNA contains two overlapping tiny (2 AAs) and small uORFs (54 AAs). To explore the role of tiny and small uORFs of SAMDC gene in controlling transcription and/or translation of downstream ORF, chimeric genes were constructed containing the point-mutated forms of the uORF sequences fused to the GUS reporter gene. In our experiment, there were a translational inhibition of its downstream GUS ORF by SAMDC uORF sequence or SAMDC uORF peptide. Expecially, translational inhibition was most effective in point-mutated construct, in which the start codon was changed. Therefore, this results suggested the ribosomal stalling might be involved in this translational inhibitory process. The frame shift in amino acid sequence of SAMDC uORF with start codon and stop codon resulted in a moderate increasing in GUS activity, suggesting the native amino acid sequence was showed more functional importance as a translational inhibitor, compared with tiny uORF sequence. Importantly, the small uORF sequence induced a real peptide of 5.7kDa, which was provided the presence of SAMDC small uORF peptide band using an in vitro transcription/translation system. Therefore, the peptide products specified by these uORFs might cause ribosomes that are translating them to stall in a sequence-dependent manner. In addition in cis repressive effect, uORF inhibited the translation of a downstream ORF in trans. The expression levels of transcripts containing uORF were markedly lower than those not containing uORF. Moreover, the half-life for degradation of the uORF-containing transcripts was also shorter. These results suggest that uORFs are sequence elements that down-regulate RNA transcripts via RNA decay mechanisms. Also, we showed that degradation rate of GUS and SAMDC protein after treatment with cycloheximide and specific proteasome inhibitor MG115 was accelerated in the presence of small uORF peptide. However, these effects of SAMDC small uORF peptide did not occurred on endogenous activity of ADC, which was other enzyme for polyamine biosynthesis and contained its own ADC uORF sequence in 5’UTR. Therefore, SAMDC small uORF has a sequence-specific function of transcriptional and/or translational inhibitor for its own SAMDC protein not only in cis but also in trans. Also, we showed that the uORF peptide specifically was phosphorylated by unknown kinase in cell-free system. Putative phosphorylation sites for protein kinase C, cAMP and cGMP dependent kinase and casein kinase Ⅱ in uORF peptide might be important for inhibitory effect of uORF. To investigate the function of the phosphorylated uORF peptide, chimeric genes were constructed containing the several phosphorylation site-mutated forms of the uORF sequences fused to the GUS reporter gene. The translational level was completely retained in phosphorylated protein of Ser17 (P17), which is a putative site for protein kinase C. Therefore, it is suggested that protein kinase C might have an important role for acting a translational inhibitor of downstream ORF. Also, RNA and protein degradation rate of downstream GUS were significantly delayed in transgenic plant without unphosphorylated small uORF than the other transgenic plants with phosphorylated uORF. These results implied that phosphorylated small uORF peptide might effectively act as a functional regulator in RNA and protein degradation. This is the first report to provide evidence that phosphorylation of small uORF may repress the translation of downstream ORF in sequence-dependent manner in plant mRNAs. Therefore it contributes to a broader understanding of the role plated by uORFs in the regulation of gene expression.
주제어
#Regulation mechanism Polyamine Protein degradation RNA degradation Translational inhibitor GUS Phosphorylation uORF SAMDC
※ AI-Helper는 부적절한 답변을 할 수 있습니다.