Functional characterization of pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle
Functional characterization of pwp2, a WD family protein essential for the assembly of the 90 S pre-ribosomal particle
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DOI：
10.1074/jbc.m404909200
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发表时间：
2004-09-03
影响因子：
4.8
通讯作者：
Bustelo, XR
中科院分区：
文献类型：
--
作者：
Dosil, M;Bustelo, XR
在这里，我们报告了Pwp2的功能特征，它是90 S前核糖体的一个进化保守成分。酵母中Pwp2蛋白的条件性缺失特异性地损害了A(0)、A(1)和A(2)位点的前rRNA加工，导致18个S rRNA和40个S核糖体亚基水平的大幅下降。前核糖体颗粒沉降分析表明，这些缺陷是由于90个S前核糖体形成受阻所致。我们证明在Pwp2缺失的细胞中，U3小核仁核糖核蛋白不能与35 S前rRNA相互作用，而是以游离复合体的形式积累。同样，在没有Pwp2的情况下，其他90个S颗粒成分如Imp3和Imp4不与前rRNA前体相关联。此外，我们还发现，在阻断U3核糖核蛋白组装后，Pwp2主要以与Dip2、Utp6、Utp13、Utp18和Utp21五种蛋白质结合的复合体形式积累。免疫沉淀和梯度沉淀分析表明，该Pwp2小亚基能够直接与35 S前rRNA5‘端相互作用。综上所述，这些结果表明，Pwp2形成了一个稳定的颗粒亚单位的一部分，独立于U3小核仁核糖核蛋白，它是90 S前核糖体初始组装步骤所必需的。
Here we report the functional characterization of Pwp2, an evolutionary conserved component of the 90 S pre-ribosome. Conditional depletion of the Pwp2 protein in yeast specifically impairs pre-rRNA proccessing at sites A(0), A(1), and A(2), leading to a strong decrease in 18 S rRNA and 40 S ribosomal subunit levels. Pre-ribosomal particle sedimentation analysis indicated that these defects are caused by a block in the formation of 90 S pre-ribosomes. We demonstrate that in Pwp2-depleted cells the U3 small nucleolar ribonucleoprotein is not able to interact with the 35 S pre-rRNA and accumulates as a free complex. Similarly, other 90 S particle components such as Imp3 and Imp4 do not associate with the pre-rRNA precursor in the absence of Pwp2. In addition, we have found that after blocking U3 ribonucleoprotein assembly, Pwp2 predominantly accumulates as a complex in association with five proteins: Dip2, Utp6, Utp13, Utp18, and Utp21. Immunoprecipitation and gradient sedimentation analysis revealed that this Pwp2 small subcomplex is capable of interacting directly with the 35 S pre-rRNA 5' end. Taken together, these results indicate that Pwp2 forms part of a stable particle subunit independent of the U3 small nucleolar ribonucleoprotein that is essential for the initial assembly steps of the 90 S pre-ribosome.