<br>通过结构-功能分析验证 EMCV IRES 二级结构,Biochemistry

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通过结构-功能分析验证 EMCV IRES 二级结构
Biochemistry ( IF 2.9 ) Pub Date : 2023-12-11 , DOI: 10.1021/acs.biochem.3c00579
Adam Maloney ₁ , Simpson Joseph ₁

Affiliation

脑心肌炎病毒内部核糖体进入位点（EMCV IRES）是在脑心肌炎病毒基因组 RNA 的 5' UTR 中发现的结构化 RNA 序列。EMCV IRES 结构有助于高效的翻译起始，无需 5′ m⁷G 帽或帽结合蛋白 eIF4E。IRES 的二级结构一直是之前几项研究的主题，并且已经提出了许多不同的结构模型。尽管 IRES 的某些域在不同的二级结构模型中是保守的，但 IRES 的域 I 在它们之间差异很大。文献比较导致确定了三个感兴趣的区域，这些区域在过去二级结构模型中显示出结构异质性。为了测试这些区域中二级结构模型的准确性，我们采用了突变分析和 SHAPE 探测。突变分析显示，已确定的感兴趣区域内的两个螺旋区域对 IRES 翻译很重要。这些螺旋区域仅与文献中的一种结构预测一致，并且在使用现代二级结构预测方法预测的 EMCV IRES 结构中未形成。在体外翻译后进行探测时，多重 SHAPE 保护进一步支持了这些区域的重要性，表明这些区域参与 IRES 翻译复合物。这项工作验证了已发布的结构，并展示了域 I 在 EMCV IRES 翻译启动过程中的重要性。

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Validating the EMCV IRES Secondary Structure with Structure–Function Analysis

The encephalomyocarditis virus internal ribosome entry site (EMCV IRES) is a structured RNA sequence found in the 5′ UTR of the genomic RNA of the encephalomyocarditis virus. The EMCV IRES structure facilitates efficient translation initiation without needing a 5′ m⁷G cap or the cap-binding protein eIF4E. The secondary structure of IRES has been the subject of several previous studies, and a number of different structural models have been proposed. Though some domains of the IRES are conserved across the different secondary structure models, domain I of the IRES varies greatly across them. A literature comparison led to the identification of three regions of interest that display structural heterogeneity within past secondary structure models. To test the accuracy of the secondary structure models in these regions, we employed mutational analysis and SHAPE probing. Mutational analysis revealed that two helical regions within the identified regions of interest are important for IRES translation. These helical regions are consistent with only one of the structure predictions in the literature and do not form in EMCV IRES structures predicted using modern secondary structure prediction methods. The importance of these regions is further supported by multiple SHAPE protections when probing was performed after in vitro translation, indicating that these regions are involved in the IRES translation complex. This work validates a published structure and demonstrates the importance of domain I during EMCV IRES translation initiation.

更新日期：2023-12-11

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