Li Lei, Wang Ching C
Department of Pharmaceutical Chemistry, University of California San Francisco, N572C, Mission Bay Genentech Hall, 600 16th St., San Francisco, CA 94122-2280, USA.
Eukaryot Cell. 2005 May;4(5):948-59. doi: 10.1128/EC.4.5.948-959.2005.
Eukaryotic translation initiation factor 4E (eIF4E) binds to the m(7)GTP of capped mRNAs and is an essential component of the translational machinery that recruits the 40S small ribosomal subunit. We describe here the identification and characterization of two eIF4E homologues in an ancient protist, Giardia lamblia. Using m(7)GTP-Sepharose affinity column chromatography, a specific binding protein was isolated and identified as Giardia eIF4E2. The other homologue, Giardia eIF4E1, bound only to the m(2,2,7)GpppN structure. Although neither homologue can rescue the function of yeast eIF4E, a knockdown of eIF4E2 mRNA in Giardia by a virus-based antisense ribozyme decreased translation, which was shown to use m(7)GpppN-capped mRNA as a template. Thus, eIF4E2 is likely the cap-binding protein in a translation initiation complex. The same knockdown approach indicated that eIF4E1 is not required for translation in Giardia. Immunofluorescence assays showed wide distribution of both homologues in the cytoplasm. But eIF4E1 was also found concentrated and colocalized with the m(2,2,7)GpppN cap, 16S-like rRNA, and fibrillarin in the nucleolus-like structure in the nucleus. eIF4E1 depletion from Giardia did not affect mRNA splicing, but the protein was bound to Giardia small nuclear RNAs D and H known to have an m(2,2,7)GpppN cap, thus suggesting a novel function not yet observed among other eIF4Es in eukaryotes.
真核生物翻译起始因子4E（eIF4E）与带帽mRNA的m（7）GTP结合，是招募40S小核糖体亚基的翻译机制的重要组成部分。我们在此描述了在一种古老的原生生物——蓝氏贾第鞭毛虫中两个eIF4E同源物的鉴定和特征。使用m（7）GTP - 琼脂糖亲和柱色谱法，分离出一种特异性结合蛋白，并鉴定为贾第鞭毛虫eIF4E2。另一个同源物，贾第鞭毛虫eIF4E1，仅与m（2,2,7）GpppN结构结合。尽管这两个同源物都不能挽救酵母eIF4E的功能，但通过基于病毒的反义核酶敲低贾第鞭毛虫中的eIF4E2 mRNA会降低翻译，这表明翻译是以m（7）GpppN带帽的mRNA为模板进行的。因此，eIF4E2可能是翻译起始复合物中的帽结合蛋白。同样的敲低方法表明，贾第鞭毛虫的翻译不需要eIF4E1。免疫荧光分析显示这两个同源物在细胞质中广泛分布。但还发现eIF4E1在细胞核中类似核仁的结构中与m（2,2,7）GpppN帽、16S样rRNA和纤维蛋白原集中并共定位。从贾第鞭毛虫中去除eIF4E1并不影响mRNA剪接，但该蛋白与已知带有m（2,2,7）GpppN帽的贾第鞭毛虫小核RNA D和H结合，因此提示了一种在真核生物其他eIF4E中尚未观察到的新功能。
