Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas - Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049, Madrid, Spain.
Sci Rep. 2018 Apr 3;8(1):5545. doi: 10.1038/s41598-018-23845-x.
Internal ribosome entry site (IRES) elements are RNA regions that recruit the translation machinery internally. Here we investigated the conformational changes and RNA dynamics of a picornavirus IRES upon incubation with distinct ribosomal fractions. Differential SHAPE analysis of the free RNA showed that nucleotides reaching the final conformation on long timescales were placed at domains 4 and 5, while candidates for long-range interactions were located in domain 3. Salt-washed ribosomes induced a fast RNA local flexibility modification of domains 2 and 3, while ribosome-associated factors changed domains 4 and 5. Consistent with this, modeling of the three-dimensional RNA structure indicated that incubation of the IRES with native ribosomes induced a local rearrangement of the apical region of domain 3, and a reorientation of domains 4 and 5. Furthermore, specific motifs within domains 2 and 3 showed a decreased flexibility upon incubation with ribosomal subunits in vitro, and presence of the IRES enhanced mRNA association to the ribosomal subunits in whole cell lysates. The finding that RNA modules can provide direct IRES-ribosome interaction suggests that linking these motifs to additional sequences able to recruit trans-acting factors could be useful to design synthetic IRESs with novel activities.
内部核糖体进入位点 (IRES) 元件是招募翻译机制的 RNA 区域。在这里，我们研究了在与不同核糖体部分孵育时，微小核糖核酸病毒 IRES 的构象变化和 RNA 动力学。游离 RNA 的差异 SHAPE 分析表明，在长时间尺度上达到最终构象的核苷酸位于结构域 4 和 5 中，而长程相互作用的候选者位于结构域 3 中。盐洗核糖体诱导结构域 2 和 3 的 RNA 局部灵活性快速修饰，而核糖体相关因子则改变结构域 4 和 5。与之一致的是，对三维 RNA 结构的建模表明，IRES 与天然核糖体孵育诱导了结构域 3 的顶端区域的局部重排，以及结构域 4 和 5 的重新定向。此外，结构域 2 和 3 内的特定基序在与体外核糖体亚基孵育时显示出灵活性降低，并且 IRES 的存在增强了在整个细胞裂解物中 mRNA 与核糖体亚基的结合。发现 RNA 模块可以提供直接的 IRES-核糖体相互作用，这表明将这些基序与能够招募反式作用因子的其他序列连接起来，可能有助于设计具有新型活性的合成 IRES。
