Translational competence of ribosomes released from a premature termination codon is modulated by NMD factors
Translational competence of ribosomes released from a premature termination codon is modulated by NMD factors
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DOI：
10.1261/rna.1987710
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发表时间：
2010-09-01
期刊：
影响因子：
4.5
通讯作者：
Jacobson, Allan
中科院分区：
文献类型：
--
作者：
Ghosh, Shubhendu;Ganesan, Robin;Jacobson, Allan
酵母UPF蛋白(UPF1、Upf2/Nmd2和Upf3)除了在促进无意义介导的mRNA衰退(NMD)中的作用外，还至少在体外表现出翻译调节功能，包括在提前翻译终止和随后的重新启动中的作用。在这里，我们发现所有的UPF Delta株在体内遇到提前终止密码子(PTC)后也无法重新启动翻译，这导致我们为所有这些翻译现象寻找一个统一的机制。比较野生型(WT)和UPF1 Delta提取物的体外翻译活性，以测试UPF1在终止后核糖体再利用中的作用。与WT提取物相比，缺乏UPF1的非核苷酸提取物对合成的CAN1/Luc mRNA的翻译活性大约降低了一倍，这一缺陷伴随着每个mRNA上的核糖体减少和启动时60S连接步骤的效率降低。这些缺陷可以通过纯化的FLAG-UPF1或60S亚基来补充，似乎反映了核糖体从内源含有PTC的mRNAs到同一提取物中外源添加的合成mRNAs的循环减少。这一假设得到了实验的验证和支持，在这些实验中，有核的WT或UPF1 Delta提取物首先用高浓度的合成mRNAs挑战，这些mRNAs是正常或过早翻译终止的模板，然后检测它们翻译正常mRNA的能力。我们的结果表明，UPF1在PTC的终止和核糖体释放与随后的核糖体再利用用于另一轮翻译起始的耦合机制中发挥关键作用。
In addition to their well-documented roles in the promotion of nonsense-mediated mRNA decay (NMD), yeast Upf proteins (Upf1, Upf2/Nmd2, and Upf3) also manifest translational regulatory functions, at least in vitro, including roles in premature translation termination and subsequent reinitiation. Here, we find that all upf Delta strains also fail to reinitiate translation after encountering a premature termination codon (PTC) in vivo, a result that led us to seek a unifying mechanism for all of these translation phenomena. Comparisons of the in vitro translational activities of wild-type (WT) and upf1 Delta extracts were utilized to test for a Upf1 role in post-termination ribosome reutilization. Relative to WT extracts, non-nucleased extracts lacking Upf1 had approximately twofold decreased activity for the translation of synthetic CAN1/LUC mRNA, a defect paralleled by fewer ribosomes per mRNA and reduced efficiency of the 60S joining step at initiation. These deficiencies could be complemented by purified FLAG-Upf1, or 60S subunits, and appeared to reflect diminished cycling of ribosomes from endogenous PTC-containing mRNAs to exogenously added synthetic mRNA in the same extracts. This hypothesis was tested, and supported, by experiments in which nucleased WT or upf1 Delta extracts were first challenged with high concentrations of synthetic mRNAs that were templates for either normal or premature translation termination and then assayed for their capacity to translate a normal mRNA. Our results indicate that Upf1 plays a key role in a mechanism coupling termination and ribosome release at a PTC to subsequent ribosome reutilization for another round of translation initiation.