The Arabidopsis AGAMOUS 5-UTR represses downstream gene translation
The Arabidopsis AGAMOUS 5-UTR represses downstream gene translation
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拟南芥 AGAMOUS 5-UTR 抑制下游基因翻译
DOI：
10.1007/s11427-018-9383-y
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Ma Ligeng
中科院分区：
文献类型：
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作者：
Cao Ying;Wang Ying;Li Yan;Yang Jing;Ma Ligeng
在真核生物中，成熟的 mRNA 具有由 5'-非翻译区 (5'-UTR)、编码区和 3'-非翻译区 (3'-UTR) 组成的三联结构。虽然编码区编码蛋白质序列，但 UTR 在控制 mRNA 翻译效率、稳定性和亚细胞定位方面发挥着至关重要的作用。 5'-UTR 的几个结构特征，包括上游起始密码子或开放阅读框 (uORF)、稳定的二级结构、内部核糖体进入位点以及与 RNA 结合蛋白相互作用的各种顺式作用元件，在调节翻译效率方面发挥着重要作用（Mignone 等，2002；Hinnebusch 等，2016；Srivastava 等，2018）。其中，从 uORF 开始翻译可以生成短肽，这些短肽能够通过核糖体停滞来抑制主 ORF 的翻译（Sachs 和 Geballe，2006；Calvo 等，2009；Hayashi 等，2017）。拟南芥基因组中5′-UTR的平均长度为155 bp；然而，编码必须精确控制其表达的发育相关蛋白的 mRNA 通常具有比平均值更长的 5'-UTR（Srivastava 等人，2018）。这使得能够产生多种 mRNA 变体并赋予翻译机制多功能性，从而帮助植物适应不断变化的环境条件（Srivastava 等人，2018）。 AGAMOUS (AG) 是拟南芥第三轮和第四轮正常发育所需的 MADS 盒转录因子，第三轮和第四轮分别产生雄蕊和心皮；它还赋予花分生组织决定性（Yanofsky et al., 1990; ÓMaoiléidigh et al., 2013; Liu et al., 2011）。根据其在花发育中的功能，AG 在花器官的第三轮和第四轮中特异性表达（Bowman 等，1991）。 AG的时空表达受到高度调控；据报道，AG 内的几种反式作用因子和基因内 DNA 元件可精确调节其转录（Lenhard 等，2001；Drews 等，1991；Riechmann 等，1999；Deyholos 和 Sieburth，2000）。此外，AG 通过前 mRNA 加工在转录后水平受到调节 (Cheng et al., 2003)。然而，对于AG翻译的控制却一无所知。 AG的5'-UTR约为500 bp，比拟南芥基因组中的平均5'-UTR长得多。因此，了解 AG 5'-UTR 是否对 AG 表达有影响将会很有趣。在本研究中，我们研究了 AG 5'-UTR 对 AG 翻译的作用。 AG mRNA 被预测有一个长度约为 500 bp 的 5'-UTR，这一预测得到了 RT-PCR 的证实（支持信息中的图 S1）。 AG 5'-UTR 连接到 GFP 上游，并在 CaMV 35S 启动子的控制下克隆到 pCAMBIA1300 中（图 1 A）。然后将所得构建体 (35S-UTR:: GFP) 与 35S:: GUS 共转染到烟草细胞中。 GUS 用作内部对照，而 GFP 则用作内部对照。
In eukaryotes, mature mRNAs have a tripartite structure consisting of a 5′-untranslated region (5′-UTR), a coding region and a 3′-untranslated region (3′-UTR). Though the coding region encodes the protein sequence, UTRs play crucial roles in the control of mRNA translation efficiency, stability and subcellular localization. Several structural features of 5′-UTRs, including upstream initiation codons or open reading frames (uORFs), stable secondary structures, internal ribosome entry sites and various cis-acting elements that interact with RNA-binding proteins, have major roles in modulating translation efficiency (Mignone et al., 2002; Hinnebusch et al., 2016; Srivastava et al., 2018). Among them, the initiation of translation from a uORF can generate short peptides that are capable of repressing translation from the main ORF by ribosome stalling (Sachs and Geballe, 2006; Calvo et al., 2009; Hayashi et al., 2017). The average length of a 5′-UTR in the Arabidopsis genome is 155 bp; however, mRNAs encoding development-related proteins whose expression must be precisely controlled often have 5′-UTRs that are longer than the average (Srivastava et al., 2018). This enables the production of multiple mRNA variants and confers versatility to the translation machinery, thereby helping plants adapt to changing environmental conditions (Srivastava et al., 2018). AGAMOUS (AG) is aMADS box transcription factor that is required in Arabidopsis for normal development of the third and fourth whorls, which give rise to stamens and carpels, respectively; it also confers determinacy to the floral meristem (Yanofsky et al., 1990; ÓMaoiléidigh et al., 2013; Liu et al., 2011). In accordance with its function in flower development, AG is specifically expressed in the third and fourth whorls of flower organs (Bowman et al., 1991). The spatiotemporal expression of AG is highly regulated; several trans-acting factors and intragenic DNA elements within AG have been reported to regulate its transcription precisely (Lenhard et al., 2001; Drews et al., 1991; Riechmann et al., 1999; Deyholos and Sieburth, 2000). In addition, AG is regulated at the posttranscriptional level through pre-mRNA processing (Cheng et al., 2003). However, nothing is known about the control of AG translation. The 5′-UTR of AG is about 500 bp, which is much longer than the average 5′-UTR in the Arabidopsis genome. Thus, it would be interesting to know whether the AG 5′-UTR has any effect on AG expression. In the present study, we investigated the role of the 5′-UTR of AG on AG translation. AG mRNAwas predicted to have a 5′-UTR about 500 bp in length, and this predication was confirmed by RT-PCR (Figure S1 in Supporting Information). AG 5′-UTR were ligated upstream of GFP and cloned into pCAMBIA1300 under the control of the CaMV 35S promoter (Figure 1 A). The resulting constructs (35S-UTR:: GFP) were then co-transfected into tobacco cells with 35S:: GUS. GUS was used as an internal control, while GFP was