Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.
Polysomes of Trypanosoma brucei: Association with Initiation Factors and RNA-Binding Proteins.
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DOI：
10.1371/journal.pone.0135973
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Clayton C
中科院分区：
文献类型：
--
作者：
Klein C;Terrao M;Inchaustegui Gil D;Clayton C
我们在这里报告的实验结果，旨在确定RNA结合蛋白，可能与布氏锥虫多核糖体。在对多聚体组分进行一些初步质谱分析后，我们使用蔗糖梯度和免疫荧光研究了选定的标记蛋白的分布。正如预期的那样，多核糖体组分包含几乎所有注释的核糖体蛋白、翻译相关蛋白质折叠复合物和许多翻译因子，而且还包含许多其他丰富的蛋白质。结果表明，帽结合蛋白EIF4E3和EIF4E4与游离和膜结合的多核糖体。存在EIF4E结合配偶体EIF4G4和EIF4G3，但未检测到其他EIF4E和EIF4G旁系同源物。多聚体组分中的显性EIF4E是EIF4E4，并且很少多聚体mRNA与EIF4G相关。在多核糖体中检测到13种潜在的mRNA结合蛋白，包括已知的多核糖体相关蛋白RBP42。在表位标记后测试了另外两种蛋白质的位置：RBP29在细胞核中，而ZC3H29在细胞质中。定量分析表明，如果蛋白质超过其靶结合位点超过25倍摩尔过量，则在蔗糖梯度中不会检测到RNA结合蛋白与多核糖体组分的特异性缔合。
We report here the results of experiments designed to identify RNA-binding proteins that might be associated with Trypanosoma brucei polysomes. After some preliminary mass spectrometry of polysomal fractions, we investigated the distributions of selected tagged proteins using sucrose gradients and immunofluorescence. As expected, the polysomal fractions contained nearly all annotated ribosomal proteins, the translation-associated protein folding complex, and many translation factors, but also many other abundant proteins. Results suggested that cap-binding proteins EIF4E3 and EIF4E4 were associated with both free and membrane-bound polysomes. The EIF4E binding partners EIF4G4 and EIF4G3 were present but the other EIF4E and EIF4G paralogues were not detected. The dominant EIF4E in the polysomal fraction is EIF4E4 and very few polysomal mRNAs are associated with EIF4G. Thirteen potential mRNA-binding proteins were detected in the polysomes, including the known polysome-associated protein RBP42. The locations of two of the other proteins were tested after epitope tagging: RBP29 was in the nucleus and ZC3H29 was in the cytoplasm. Quantitative analyses showed that specific association of an RNA-binding protein with the polysome fraction in sucrose gradients will not be detected if the protein is in more than 25-fold molar excess over its target binding sites.