Structure of yeast U6 snRNPs: Arrangement of Prp24p and the LSm complex as revealed by electron microscopy
Structure of yeast U6 snRNPs: Arrangement of Prp24p and the LSm complex as revealed by electron microscopy
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DOI：
10.1261/rna.1369808
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发表时间：
2008-12-01
期刊：
影响因子：
4.5
通讯作者：
Luehrmann, Reinhard
中科院分区：
文献类型：
--
作者：
Karaduman, Ramazan;Dube, Prakash;Luehrmann, Reinhard
U6 snRNP的蛋白质组分（Prp 24 p和LSm 2 -8）被认为在U4/U6 di-snRNP形成期间协同作用以促进U6和U4 snRNA的退火。为了进一步了解这些蛋白在S.酿酒酵母U6 snRNP，我们研究了这种颗粒的结构，通过电子显微镜。U6 snRNP通过亲和层析和梯度离心纯化，然后立即吸附到碳膜支持物上，显示出开放形式，其中Prp 24蛋白和由LSm蛋白形成的环作为两个独立的形态结构域可见，而在结合到碳膜之前在温和条件下通过溶液中的化学交联稳定的颗粒表现出紧凑形式，两个域彼此非常接近。在开放的形式，个别LSm蛋白定位的一种新的方法，采用C-末端基因标记的LSm蛋白与yECitrine。这些研究显示Prp 24蛋白与LSm环的每个亚基的距离是确定的，这反过来表明LSm环以一致的方式定位在U6 RNA上。此外，与EM观察结果一致，UV交联显示U6 RNA在Prp 24 p和LSm环之间的界面处与LSm 2蛋白接触。此外，LSmp-Prp 24 p相互作用可能限于封闭形式，这似乎代表了U6 snRNP颗粒的溶液结构。
Protein components of the U6 snRNP (Prp24p and LSm2-8) are thought to act cooperatively in facilitating the annealing of U6 and U4 snRNAs during U4/U6 di-snRNP formation. To learn more about the spatial arrangement of these proteins in S. cerevisiae U6 snRNPs, we investigated the structure of this particle by electron microscopy. U6 snRNPs, purified by affinity chromatography and gradient centrifugation, and then immediately adsorbed to the carbon film support, revealed an open form in which the Prp24 protein and the ring formed by the LSm proteins were visible as two separate morphological domains, while particles stabilized by chemical cross-linking in solution under mild conditions before binding to the carbon film exhibited a compact form, with the two domains in close proximity to one another. In the open form, individual LSm proteins were located by a novel approach employing C-terminal genetic tagging of the LSm proteins with yECitrine. These studies show the Prp24 protein at defined distances from each subunit of the LSm ring, which in turn suggests that the LSm ring is positioned in a consistent manner on the U6 RNA. Furthermore, in agreement with the EM observations, UV cross-linking revealed U6 RNA in contact with the LSm2 protein at the interface between Prp24p and the LSm ring. Further, LSmp-Prp24p interactions may be restricted to the closed form, which appears to represent the solution structure of the U6 snRNP particle.