Molecular mapping of the determinants involved in human Staufen-ribosome association
Molecular mapping of the determinants involved in human Staufen-ribosome association
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DOI：
10.1042/bj20020263
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发表时间：
2002-08-01
影响因子：
4.1
通讯作者：
DesGroseillers, L
中科院分区：
文献类型：
--
作者：
Luo, M;Duchaîne, TF;DesGroseillers, L
人双链（ds）RNA结合蛋白Staufen（hStau）被认为在RNA转运及其定位中起作用。通过使用蔗糖梯度沉降分析，我们发现表观分子量为55 kDa的Staufen异构体（Stau（55））与核糖体共分级分离，并与40和60 S核糖体亚基结合，表明Staufen异构体hStau（55）在翻译中起一定作用。为了绘制参与这种关联的决定簇，我们产生了一系列缺失突变体，并通过细胞分级分离和荧光免疫显微镜分析了它们的亚细胞分布。我们的研究结果表明，多个决定因素促进hStau（55）-核糖体协会通过RNA结合依赖性机制和蛋白质-蛋白质相互作用。RNA结合蛋白结构域3（dsRBD 3）的RNA结合活性而不是dsRBD 4的RNA结合活性是第一决定因素。尽管与核糖体的稳定结合是必需的，但单独的dsRBD 3是不够的，需要其他决定因素作为辅助因子。一致地，当dsRBD 4和微管蛋白结合结构域一起表达时，其构成最小的Stau（55）/核糖体蛋白-蛋白缔合结构域。Stau（55）的这一区域足以独立地与核糖体缔合，但需要dsRBD 3的RNA结合活性才能完全缔合。因此，结果与Stau（55）在翻译调节中的假定作用一致。
The human double-stranded (ds) RNA-binding protein Staufen (hStau) is considered to have a role in RNA transport and its localization. By using sedimentation analysis on sucrose gradients, we showed that the Staufen isoform with an apparent molecular mass of 55 kDa (Stau(55)) co-fractionated with ribosomes and associated with both the 40 and 60 S ribosomal subunits, suggesting that the Staufen isoform hStau(55) plays some role in translation. To map the determinant(s) involved in this association, we generated a series of deletion mutants and analysed their subcellular distribution by cell fractionation and fluorescent immunomicroscopy. Our results demonstrated that multiple determinants promote hStau(55)-ribosome association via both an RNA-binding-dependent mechanism and protein-protein interaction. The RNA-binding activity of the RNA-binding protein domain 3 (dsRBD3) but not that of dsRBD4 is the first determinant. Although necessary for stable association with ribosomes, dsRBD3 alone is not sufficient and needs other determinants as co-factors. Consistently, when expressed together, dsRBD4 and the tubulin-binding domain constitute the minimal Stau(55)/ribosome protein-protein association domain. This region of Stau(55) is sufficient to associate with ribosomes independently, but requires the RNA-binding activity of dsRBD3 for complete association. Thus the results are consistent with a putative role for Stau(55) in the regulation of translation.