Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels
Genome-wide identification of Arabidopsis non-AUG-initiated upstream ORFs with evolutionarily conserved regulatory sequences that control protein expression levels
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DOI：
10.1007/s11103-022-01309-1
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发表时间：
2021-03
影响因子：
5.1
通讯作者：
Yuta Hiragori;Hiro Takahashi;Noriya Hayashi;Shun-ichi Sasaki;Kodai Nakao;Taichiro Motomura;Y. Yamashita;S. Naito;H. Onouchi
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文献类型：
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作者：
Yuta Hiragori;Hiro Takahashi;Noriya Hayashi;Shun-ichi Sasaki;Kodai Nakao;Taichiro Motomura;Y. Yamashita;S. Naito;H. Onouchi
关键信息本研究在拟南芥中发现了4个新的具有进化保守序列的非AUG启动的上游ORF（uORF），并阐明了非AUG启动的uORF促进主ORF翻译的机制。最近的全基因组核糖体分析研究表明，数百或数千个uORF在非AUG起始密码子处启动翻译。然而，这些非AUG uORF的生理学意义迄今为止仅在其中少数几个中得到证实。本研究采用生物信息学和实验分析相结合的方法，对拟南芥中具有生理学意义的非AUG uORF进行了鉴定。由于生理上重要的非AUG uORF很可能在物种间保守，我们首先在拟南芥基因组中搜索具有进化保守序列的非AUG启动的uORF。然后，我们使用瞬时表达测定来检查保守的非AUG uORF对下游mORF表达的影响。结果，鉴定出三个抑制性和一个促进性非AUG uORF。在抑制性非AUG uORF中，有两个以氨基酸序列依赖的方式对mORF表达产生抑制作用。这两个非AUG uORF可能编码引起核糖体停滞的调节肽，从而增强其抑制作用。相比之下，其中一个鉴定的调节性非AUG uORF通过减轻下游AUG起始uORF的抑制作用来促进mORF表达。这些发现提供了深入了解的机制，使非AUG uORF发挥调控作用，尽管他们的翻译起始效率低。
Key messageThis study identified four novel regulatory non-AUG-initiated upstream ORFs (uORFs) with evolutionarily conserved sequences in Arabidopsis and elucidated the mechanism by which a non-AUG-initiated uORF promotes main ORF translation.AbstractUpstream open reading frames (uORFs) are short ORFs found in the 5′-untranslated regions (5′-UTRs) of eukaryotic transcripts and can influence the translation of protein-coding main ORFs (mORFs). Recent genome-wide ribosome profiling studies have revealed that hundreds or thousands of uORFs initiate translation at non-AUG start codons. However, the physiological significance of these non-AUG uORFs has so far been demonstrated for only a few of them. In this study, to identify physiologically important regulatory non-AUG uORFs inArabidopsis, we took an approach that combined bioinformatics and experimental analysis. Since physiologically important non-AUG uORFs are likely to be conserved across species, we first searched theArabidopsisgenome for non-AUG-initiated uORFs with evolutionarily conserved sequences. Then, we examined the effects of the conserved non-AUG uORFs on the expression of the downstream mORFs using transient expression assays. As a result, three inhibitory and one promotive non-AUG uORFs were identified. Among the inhibitory non-AUG uORFs, two exerted repressive effects on mORF expression in an amino acid sequence-dependent manner. These two non-AUG uORFs are likely to encode regulatory peptides that cause ribosome stalling, thereby enhancing their repressive effects. In contrast, one of the identified regulatory non-AUG uORFs promoted mORF expression by alleviating the inhibitory effect of a downstream AUG-initiated uORF. These findings provide insights into the mechanisms that enable non-AUG uORFs to play regulatory roles despite their low translation initiation efficiencies.