Molecular genetic analysis and screen for nascent-peptide mediated regulation in Arabidopsis

在对编码甲硫氨酸生物合成关键酶的拟南芥CGS1基因转录后调控的研究中，我们发现新生的CGS1多肽对蛋氨酸的代谢物S-腺苷蛋氨酸的反应介导了自身的翻译延伸停滞。虽然在真核生物中已经报道了一些由新生多肽介导的核糖体调节的例子，但除了CGS1基因外，所有其他调节新生多肽的基因都是由位于5‘非编码区的上游ORF(UORF)编码的。在这些系统中，uORF编码的多肽导致核糖体停滞，导致主要ORF的翻译下调。为了寻找新的受新生多肽调控的拟南芥基因，我们从拟南芥全长cDNA数据库中筛选出含有uORF的基因，并用拟南芥原生质体瞬时检测它们对主要ORF翻译的影响的氨基酸序列依赖性。从这个筛查中，一个uORF的移码突变被发现导致主要ORF的上调。丙氨酸扫描分析表明，该uORF编码的多肽的C-末端部分对调节很重要。相反，另一个uORF的移码突变导致了主要ORF的下调。

In the course of study on the posttranscriptional regulation of the Arabidopsis CGS1 gene, which encodes the key enzyme of methionine biosynthesis, we have found that the nascent CGS1 peptide mediates its own translation elongation arrest in response to S-adenosylmethionine, a metabolite of methionine. Although several examples of nascent peptide-mediated ribosomal regulations have been reported in eukaryotes, all other regulatory nascent peptides are encoded by upstream ORFs (uORF) located in the 5'UTR, except for the CGS1 gene. In these systems, uORF-encoded peptides cause ribosome stalling, resulting in downregulation of translation of the main ORF.In order to identify new Arabidopsis genes that are regulated by nascent peptide, we screened for uORF-containing genes from Arabidopsis full-length cDNA database, and tested their amino acid-sequence dependency of the effect on translation of the main ORF by using transient assay with Arabidopsis protoplasts. From this screen, a frameshift mutation of one uORF was found to cause upregulation of the main ORF. Alanine scanning analysis showed that C-terminal part of the peptide encoded by this uORF is important for the regulation. In contrast, a frameshift mutation of another uORF caused downregulation of the main ORF.