Institute of Biochemistry and Biotechnology and Charles Tanford Protein Center, Martin Luther University Halle-Wittenberg, 06099 Halle, Germany.
RNA. 2020 May;26(5):613-628. doi: 10.1261/rna.073759.119. Epub 2020 Feb 28.
Functions of eukaryotic mRNAs are characterized by intramolecular interactions between their ends. We have addressed the question whether 5' and 3' ends meet by diffusion-controlled encounter "through solution" or by a mechanism involving the RNA backbone. For this purpose, we used a translation system derived from embryos that displays two types of 5'-3' interactions: Cap-dependent translation initiation is stimulated by the poly(A) tail and inhibited by Smaug recognition elements (SREs) in the 3' UTR. Chimeric RNAs were made consisting of one RNA molecule carrying a luciferase coding sequence and a second molecule containing SREs and a poly(A) tail; the two were connected via a protein linker. The poly(A) tail stimulated translation of such chimeras even when disruption of the RNA backbone was combined with an inversion of the 5'-3' polarity between the open reading frame and poly(A) segment. Stimulation by the poly(A) tail also decreased with increasing RNA length. Both observations suggest that contacts between the poly(A) tail and the 5' end are established through solution, independently of the RNA backbone. In the same chimeric constructs, SRE-dependent inhibition of translation was also insensitive to disruption of the RNA backbone. Thus, tracking of the backbone is not involved in the repression of cap-dependent initiation. However, SRE-dependent repression was insensitive to mRNA length, suggesting that the contact between the SREs in the 3' UTR and the 5' end of the RNA might be established in a manner that differs from the contact between the poly(A) tail and the cap.
真核生物 mRNA 的功能特征是其两端分子内的相互作用。我们提出了一个问题，即 5' 和 3' 端是否通过扩散控制的“通过溶液”相遇，还是通过涉及 RNA 主链的机制相遇。为此，我们使用了一种源自胚胎的翻译系统，该系统显示了两种 5'-3' 相互作用类型：多聚（A）尾刺激帽依赖性翻译起始，而 Smaug 识别元件（SREs）在 3'UTR 中抑制翻译。嵌合 RNA 由一个携带荧光素酶编码序列的 RNA 分子和第二个含有 SREs 和多聚（A）尾的 RNA 分子组成；这两个分子通过蛋白质接头连接。当 RNA 主链的破坏与开放阅读框和多聚（A）片段之间的 5'-3' 极性的反转相结合时，多聚（A）尾刺激这种嵌合 RNA 的翻译。多聚（A）尾的刺激作用也随着 RNA 长度的增加而降低。这两个观察结果表明，多聚（A）尾和 5' 端之间的接触是通过溶液建立的，而与 RNA 主链无关。在相同的嵌合构建体中，SRE 依赖性翻译抑制也不受 RNA 主链破坏的影响。因此，主链的跟踪不参与帽依赖性起始的抑制。然而，SRE 依赖性抑制对 mRNA 长度不敏感，这表明 3'UTR 中的 SREs 和 RNA 5' 端之间的接触可能以不同于多聚（A）尾和帽之间的接触的方式建立。
