Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid, Spain.
RNA. 2010 Apr;16(4):852-62. doi: 10.1261/rna.1973710. Epub 2010 Mar 1.
Translation initiation driven by internal ribosome entry site (IRES) elements is dependent on the structural organization of the IRES region. We have previously shown that a structural motif within the foot-and-mouth-disease virus IRES is recognized in vitro as substrate for the Synechocystis sp. RNase P ribozyme. Here we show that this structure-dependent endonuclease recognizes the IRES element in cultured cells, leading to inhibition of translation. Inhibition of IRES activity was dependent on the expression of the active ribozyme RNA subunit. Moreover, expression of the antisense sequence of the ribozyme did not inhibit IRES activity, demonstrating that stable RNA structures located upstream of the IRES element do not interfere with internal initiation. RNAs carrying defective IRES mutants that were substrates of the ribozyme in vivo revealed an increased translation of the reporter in response to the expression of the active ribozyme. In support of RNA cleavage, subsequent analysis of the translation initiation manner indicated a switch from IRES-dependent to 5'-end-dependent translation of RNase P target RNAs. We conclude that the IRES element is inactivated by expression in cis of RNase P in the cytoplasm of cultured cells, providing a promising antiviral tool to combat picornavirus infections. Furthermore, our results reinforce the essential role of the structural motif that serves as RNase P recognition motif for IRES activity.
翻译起始由内部核糖体进入位点 (IRES) 元件驱动，这取决于 IRES 区域的结构组织。我们之前已经表明，口蹄疫病毒 IRES 内的结构基序在体外被鉴定为 Synechocystis sp. RNase P 核酶的底物。在这里，我们表明这种结构依赖性内切核酸酶在培养细胞中识别 IRES 元件，导致翻译抑制。IRES 活性的抑制依赖于活性核酶 RNA 亚基的表达。此外，核酶反义序列的表达不会抑制 IRES 活性，这表明位于 IRES 元件上游的稳定 RNA 结构不会干扰内部起始。携带体内核酶底物缺陷 IRES 突变体的 RNA 显示出对活性核酶表达的反应中报告基因翻译的增加。为了支持 RNA 切割，随后对翻译起始方式的分析表明，从 IRES 依赖性到 5'-端依赖性翻译的转变发生在 RNase P 靶 RNA 上。我们得出结论，IRES 元件在培养细胞的细胞质中顺式表达 RNase P 而失活，为对抗小核糖核酸病毒感染提供了一种有前途的抗病毒工具。此外，我们的结果强化了作为 IRES 活性 RNase P 识别基序的结构基序的重要作用。
