A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity
A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity
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DOI：
10.1073/pnas.97.4.1536
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发表时间：
2000-02-15
影响因子：
11.1
通讯作者：
Mauro, VP
中科院分区：
文献类型：
--
作者：
Chappell, SA;Edelman, GM;Mauro, VP
这项研究解决了同源结构域蛋白 Ctx mRNA 中新发现的内部核糖体进入位点 (IRES) 的特性，从任一端连续删除 5' 非翻译区 (UTR) 并没有定义明显的 IRES 边界；当测试五个不重叠的 UTR 片段时，四个具有 IRES 活性。这些观察结果与其他细胞 IRES 分析一致，表明一些细胞 IRES 由独立和组合地贡献整体 IRES 活性的片段（IRES 模块）组成。我们表征了来自 Ctx 5' UTR 的 9-nt IRES 模块，该模块在核苷酸 1132-1124 处与 18S rRNA 100% 互补。在之前的工作中，我们证明该 mRNA 片段可以与其完整的 40S 亚基内的补体交联。在这里，我们表明，增加双顺反子 mRNA 顺反子间区域中该 IRES 模块的拷贝数可强烈增强各种细胞中的 IRES 活性。线。 Neuro 2a 细胞中 10 个连接的拷贝将 IRES 活性提高了 570 倍。在相同的测定系统中进行测试时，这种 IRES 活性水平比使用已充分表征的脑心肌炎病毒 IRES 获得的活性高出 63 倍。当两个 9-nt Ctx IRES 模块之间的核苷酸数量增加时，它们之间的协同作用就会降低。根据这些发现，我们讨论了细胞 mRNA 募集核糖体的可能机制，解决了高阶 RNA 结构对细胞 IRES 活性的拟议作用，并提出了 IRES 模块和转录增强子元件之间的相似之处。
This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Ctx, Sequential deletions of the 5' untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Ctx 5' UTR that is 100% complementary to the 18S rRNA at nucleotides 1132-1124, In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits, Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Ctx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.