Department of Cell Biology and Molecular Genetics, University of Maryland - College Park, College Park, MD 20742, USA.
Basic Science Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.
Nucleic Acids Res. 2022 Feb 22;50(3):1601-1619. doi: 10.1093/nar/gkac007.
Canonical eukaryotic mRNA translation requires 5'cap recognition by initiation factor 4E (eIF4E). In contrast, many positive-strand RNA virus genomes lack a 5'cap and promote translation by non-canonical mechanisms. Among plant viruses, PTEs are a major class of cap-independent translation enhancers located in/near the 3'UTR that recruit eIF4E to greatly enhance viral translation. Previous work proposed a single form of PTE characterized by a Y-shaped secondary structure with two terminal stem-loops (SL1 and SL2) atop a supporting stem containing a large, G-rich asymmetric loop that forms an essential pseudoknot (PK) involving C/U residues located between SL1 and SL2. We found that PTEs with less than three consecutive cytidylates available for PK formation have an upstream stem-loop that forms a kissing loop interaction with the apical loop of SL2, important for formation/stabilization of PK. PKs found in both subclasses of PTE assume a specific conformation with a hyperreactive guanylate (G*) in SHAPE structure probing, previously found critical for binding eIF4E. While PTE PKs were proposed to be formed by Watson-Crick base-pairing, alternative chemical probing and 3D modeling indicate that the Watson-Crick faces of G* and an adjacent guanylate have high solvent accessibilities. Thus, PTE PKs are likely composed primarily of non-canonical interactions.
真核生物的 mRNA 翻译需要起始因子 4E（eIF4E）识别 5'帽。相比之下，许多正链 RNA 病毒基因组缺乏 5'帽，并通过非典型机制促进翻译。在植物病毒中，PTE 是一类主要的帽非依赖性翻译增强子，位于/靠近 3'UTR，招募 eIF4E 以极大地增强病毒翻译。以前的工作提出了一种单一形式的 PTE，其特征是具有两个末端茎环（SL1 和 SL2）的 Y 型二级结构，顶部是一个包含大的富含 G 的不对称环的支撑茎，该环形成一个必需的假结（PK），涉及位于 SL1 和 SL2 之间的 C/U 残基。我们发现，具有少于三个连续可用的 Cyt 用于 PK 形成的 PTEs 具有一个上游茎环，该茎环与 SL2 的顶端环形成亲吻环相互作用，这对于 PK 的形成/稳定很重要。在两类 PTE 中发现的 PK 都采用了特定的构象，在 SHAPE 结构探测中具有超反应性的鸟苷（G*），这以前被发现对于结合 eIF4E 至关重要。虽然 PTE PK 被提议通过 Watson-Crick 碱基配对形成，但替代化学探测和 3D 建模表明 G*和相邻鸟苷的 Watson-Crick 面具有高溶剂可及性。因此，PTE PK 可能主要由非典型相互作用组成。
