Yoffe Yael, Zuberek Joanna, Lerer Asaf, Lewdorowicz Magdalena, Stepinski Janusz, Altmann Michael, Darzynkiewicz Edward, Shapira Michal
Department of Life Sciences, Ben Gurion University, P.O. Box 653, Beer Sheva 84105, Israel.
Eukaryot Cell. 2006 Dec;5(12):1969-79. doi: 10.1128/EC.00230-06. Epub 2006 Oct 13.
The 5' cap structure of trypanosomatid mRNAs, denoted cap 4, is a complex structure that contains unusual modifications on the first four nucleotides. We examined the four eukaryotic initiation factor 4E (eIF4E) homologues found in the Leishmania genome database. These proteins, denoted LeishIF4E-1 to LeishIF4E-4, are located in the cytoplasm. They show only a limited degree of sequence homology with known eIF4E isoforms and among themselves. However, computerized structure prediction suggests that the cap-binding pocket is conserved in each of the homologues, as confirmed by binding assays to m(7)GTP, cap 4, and its intermediates. LeishIF4E-1 and LeishIF4E-4 each bind m(7)GTP and cap 4 comparably well, and only these two proteins could interact with the mammalian eIF4E binding protein 4EBP1, though with different efficiencies. 4EBP1 is a translation repressor that competes with eIF4G for the same residues on eIF4E; thus, LeishIF4E-1 and LeishIF4E-4 are reasonable candidates for serving as translation factors. LeishIF4E-1 is more abundant in amastigotes and also contains a typical 3' untranslated region element that is found in amastigote-specific genes. LeishIF4E-2 bound mainly to cap 4 and comigrated with polysomal fractions on sucrose gradients. Since the consensus eIF4E is usually found in 48S complexes, LeishIF4E-2 could possibly be associated with the stabilization of trypanosomatid polysomes. LeishIF4E-3 bound mainly m(7)GTP, excluding its involvement in the translation of cap 4-protected mRNAs. It comigrates with 80S complexes which are resistant to micrococcal nuclease, but its function is yet unknown. None of the isoforms can functionally complement the Saccharomyces cerevisiae eIF4E, indicating that despite their structural conservation, they are considerably diverged.
锥虫mRNA的5'帽结构（称为帽4）是一种复杂结构，在前四个核苷酸上含有异常修饰。我们研究了利什曼原虫基因组数据库中发现的四种真核起始因子4E（eIF4E）同源物。这些蛋白，称为利什曼原虫IF4E-1至利什曼原虫IF4E-4，位于细胞质中。它们与已知的eIF4E异构体之间以及它们彼此之间仅表现出有限程度的序列同源性。然而，计算机化结构预测表明，帽结合口袋在每个同源物中都是保守的，这通过与m（7）GTP、帽4及其中间体的结合试验得到证实。利什曼原虫IF4E-1和利什曼原虫IF4E-4与m（7）GTP和帽4的结合效果相当好，并且只有这两种蛋白能够与哺乳动物eIF4E结合蛋白4EBP1相互作用，尽管效率不同。4EBP1是一种翻译抑制因子，与eIF4G竞争eIF4E上的相同残基；因此，利什曼原虫IF4E-1和利什曼原虫IF4E-4是作为翻译因子的合理候选者。利什曼原虫IF4E-1在无鞭毛体中更为丰富，并且还包含在无鞭毛体特异性基因中发现的典型3'非翻译区元件。利什曼原虫IF4E-2主要与帽4结合，并在蔗糖梯度上与多核糖体组分一起迁移。由于共有eIF4E通常存在于48S复合物中，利什曼原虫IF4E-2可能与锥虫多核糖体的稳定有关。利什曼原虫IF4E-3主要结合m（7）GTP，排除了其参与帽4保护的mRNA翻译的可能性。它与对微球菌核酸酶有抗性的80S复合物一起迁移，但其功能尚不清楚。这些异构体均不能在功能上互补酿酒酵母eIF4E，这表明尽管它们在结构上保守，但它们在很大程度上已经分化。
