Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
Nucleic Acids Res. 2011 Oct;39(19):8572-85. doi: 10.1093/nar/gkr560. Epub 2011 Jul 8.
RNA structure plays a fundamental role in internal initiation of translation. Picornavirus internal ribosome entry site (IRES) are long, efficient cis-acting elements that recruit the ribosome to internal mRNA sites. However, little is known about long-range constraints determining the IRES RNA structure. Here, we sought to investigate the functional and structural relevance of the invariant apical stem of a picornavirus IRES. Mutation of this apical stem revealed better performance of G:C compared with C:G base pairs, demonstrating that the secondary structure solely is not sufficient for IRES function. In turn, mutations designed to disrupt the stem abolished IRES activity. Lack of tolerance to accept genetic variability in the apical stem was supported by the presence of coupled covariations within the adjacent stem-loops. SHAPE structural analysis, gel mobility-shift and microarrays-based RNA accessibility revealed that the apical stem contributes to maintain IRES RNA structure through the generation of distant interactions between two adjacent stem-loops. Our results demonstrate that a highly interactive structure constrained by distant interactions involving invariant G:C base pairs plays a key role in maintaining the RNA conformation necessary for IRES-mediated translation.
RNA 结构在翻译的内部起始中起着至关重要的作用。微小 RNA 内部核糖体进入位点 (IRES) 是长而有效的顺式作用元件，可将核糖体募集到内部 mRNA 位点。然而，关于决定 IRES RNA 结构的长程约束的信息知之甚少。在这里，我们试图研究一种微小 RNA IRES 的不变的顶端茎的功能和结构相关性。该顶端茎的突变显示 G:C 比 C:G 碱基对具有更好的性能，这表明二级结构本身不足以满足 IRES 的功能。反过来，设计用于破坏茎的突变会使 IRES 失去活性。顶端茎中不能接受遗传变异的缺乏耐受性得到了相邻茎环内耦合变异性的支持。SHAPE 结构分析、凝胶迁移率变动和基于微阵列的 RNA 可及性揭示，顶端茎通过在两个相邻的茎环之间产生远距离相互作用，有助于维持 IRES RNA 结构。我们的结果表明，一种由涉及不变的 G:C 碱基对的远距离相互作用约束的高度相互作用的结构在维持 IRES 介导的翻译所需的 RNA 构象方面起着关键作用。
