eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA
eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA
复制标题
DOI：
10.1101/gad.480508
复制
发表时间：
2008-09-01
影响因子：
10.5
通讯作者：
Valasek, Leos
中科院分区：
文献类型：
--
作者：
Szamecz, Bela;Rutkai, Edit;Valasek, Leos
酵母起始因子eIF3（真核起始因子3）参与了翻译起始的多个步骤。先前，我们发现eIF3a的n端结构域（NTD）与位于mRNA出口通道附近的小核糖体蛋白RPS0A相互作用，eIF3可能位于mRNA出口通道附近。在这里，我们证明了eIF3a的rps0a结合域的部分缺失会损害翻译起始，并减少eIF3和相关的eIFs与体内天然起始前复合物的结合。引人注目的是，它还严重阻断了通过重启动发生的GCN4翻译的诱导。详细的检查揭示了一种新的再启动缺陷，这是由于40S核糖体在第一个上游ORF终止后无法恢复扫描（uORF1）。遗传分析揭示了eIF3a-NTD与uORF1序列5 '之间的功能相互作用，这对于增强再启动至关重要。我们进一步证明，这些刺激序列必须相对于uORF1终止密码子精确定位，并且uORF1后的再启动效率随着其长度的增加而下降。总之，我们的研究结果表明，eIF3在整个uORF1翻译过程中保留在核糖体上，并且在终止后，与mRNA出口通道的5 '增强子相互作用，以稳定mRNA与终止后40S亚基的关联，并使下游重新启动的扫描恢复。
Yeast initiation factor eIF3 ( eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5 ' of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5 ' enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.