Translation initiation factor eIF4F is essential for cap-dependent translation initiation in eukaryotes. eIF4F is a trimeric complex consisting of a scaffold protein eIF4G, cap-binding protein eIF4E and DEAD-box RNA helicase eIF4A. eIF4F binds to the 5’ cap structure of the mRNA through eIF4E and facilitates the binding of the preinitiation complex (PIC) via protein-protein interactions of eIF4G with eIF3 in mammals or with eIF5 in yeast. Initiation factor eIF4A is known to unwind the secondary structures of the 5’UTRs encountered by the PIC during its initial binding to the mRNA and while scanning for the initiation codon. In Giardia, homologs for eIF4E (GleIF4E2) and eIF4A (GleIF4A) have been identified but not for eIF4G. To address how PIC is recruited to the 5’ end of mRNA in the absence of eIF4G homolog, we have used yeast two-hybrid assays to identify potential interactions of GleIF4E2 with the components of the PIC. The results show that GleIF4E2 can interact with the β subunit of the initiation factor GleIF2, a component of the PIC. ZDOCK modeling of the GleIF4E2-GleIF2β complex revealed that the dorsal side of GleIF4E2 is likely involved in binding to GleIF2β, which mimics the interaction of mammalian eIF4E with eIF4G, and with eIF4E binding proteins. These results suggest that GleIF4E2 can facilitate the recruitment of the PIC to the 5’end of the mRNA by binding directly to the components of the PIC. The role of GleIF4A in translation initiation in Giardia is not clearly understood as the short 5’ UTRs of the mRNA are unlikely to form secondary structures. Interestingly, Pateamine A, a specific inhibitor of human eIF4A, inhibited the growth of Giardia in a dose-dependent manner, suggesting that the activity of GleIF4A is probably required for translation. Using yeast two-hybrid assays, we have identified a novel interaction of GleIF4A with i subunit of the initiation factor GleIF3 (GleIF3i), another component of the PIC. These results indicate that the GleIF4A can also interact directly with the components of the PIC. ZDOCK modeling of the GleIF3i-GleIF4A complex suggests that GleIF3i could serve as a stimulator of GleIF4A activity.

翻译起始因子eIF4F对于真核生物中依赖于帽的翻译起始是必不可少的。eIF4F是由支架蛋白eIF4G，帽结合蛋白eIF4E和DEAD-box RNA解旋酶eIF4A组成的三聚体复合物。eIF4F通过eIF4E与mRNA的5'帽结构结合，并通过哺乳动物中的eIF4G与eIF3或酵母中的eIF5的蛋白质​​-蛋白质相互作用，促进预起始复合物（PIC）的结合。已知起始因子eIF4A可以解开PIC在其最初与mRNA结合期间以及扫描起始密码子时所遇到的5'UTR的二级结构。在贾第虫，已鉴定出eIF4E（GleIF4E2）和eIF4A（GleIF4A）的同源物，但未鉴定eIF4G的同源物。为了解决在没有eIF4G同源物的情况下如何将PIC募集到mRNA的5'端，我们使用了酵母双杂交测定法来鉴定GleIF4E2与PIC组件的潜在相互作用。结果表明，GleIF4E2可以与PIC的起始因子GleIF2的β亚基相互作用。对GleIF4E2-GleIF2β复合体的ZDOCK建模表明，GleIF4E2的背侧可能参与了与GleIF2β的结合，从而模仿了哺乳动物eIF4E与eIF4G和与eIF4E结合蛋白的相互作用。这些结果表明，GleIF4E2可通过直接与PIC的成分结合而促进PIC募集到mRNA的5'末端。GleIF4A在翻译起始中的作用贾第鞭毛虫尚不清楚，因为mRNA的短5'UTR不太可能形成二级结构。有趣的是，人eIF4A的一种特异性抑制剂三胺A以剂量依赖性的方式抑制了贾第鞭毛虫的生长，这表明翻译可能需要GleIF4A的活性。使用酵母双杂交测定法，我们确定了GleIF4A与PIC另一成分起始因子GleIF3（GleIF3i）的i亚基的新型相互作用。这些结果表明，GleIF4A也可以直接与PIC的组件进行交互。GleIF3i-GleIF4A复合体的ZDOCK建模表明，GleIF3i可以作为GleIF4A活性的刺激物。