Control of mammalian translation by mRNA structure near caps
Control of mammalian translation by mRNA structure near caps
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DOI：
10.1261/rna.2309906
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发表时间：
2006-05-01
期刊：
影响因子：
4.5
通讯作者：
Tsien, RY
中科院分区：
文献类型：
--
作者：
Babendure, JR;Babendure, JL;Tsien, RY
RNA翻译的扫描模型提出，mRNA中高度稳定的二级结构可以抑制翻译，而如果足够接近5'甲基G帽，则较低热稳定性的结构也会影响翻译。然而，只有零碎的信息是可用的依赖于在活的哺乳动物细胞中的翻译效率的热力学稳定性，位置，和GC含量的RNA结构的5 '-非翻译区。我们设计了一种用于单个活细胞中翻译效率的双色荧光测定法，并比较了预测热稳定性范围为-10至-50 kcal/mol且5 'G帽-发夹距离为1-46个碱基的各种发夹。当发夹稳定性从Δ G = -25增加到-35 kcal/mol时，翻译效率突然降低。相对于5 - 9帽，移动发夹小至9个碱基可以调节翻译超过50倍。当预测的热稳定性和帽-发夹距离保持恒定时，增加GC含量降低了翻译效率。我们还发现，与体外裂解物中的翻译相比，天然存在的5 '-非翻译区在活细胞中对翻译的影响不同。我们的研究将帮助科学家设计实验，故意调节哺乳动物翻译与设计的5'UTR。
The scanning model of RNA translation proposes that highly stable secondary structures within mRNAs can inhibit translation, while structures of lower thermal stability also affect translation if close enough to the 5' methyl G cap. However, only fragmentary information is available about the dependence of translation efficiency in live mammalian cells on the thermodynamic stability, location, and GC content of RNA structures in the 5'-untranslated region. We devised a two-color fluorescence assay for translation efficiency in single live cells and compared a wide range of hairpins with predicted thermal stabilities ranging from -10 to -50 kcal/mol and 5'G cap-to-hairpin distances of 1-46 bases. Translation efficiency decreased abruptly as hairpin stabilities increased from Delta G = -25 to -35 kcal/mol. Shifting a hairpin as little as nine bases relative to the 5 9 cap could modulate translation more than 50-fold. Increasing GC content diminished translation efficiency when predicted thermal stability and cap-to-hairpin distances were held constant. We additionally found naturally occurring 5'-untranslated regions affected translation differently in live cells compared with translation in in vitro lysates. Our study will assist scientists in designing experiments that deliberately modulate mammalian translation with designed 5' UTRs.