Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain
Nob1 binds the single-stranded cleavage site D at the 3′-end of 18S rRNA with its PIN domain
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DOI：
10.1073/pnas.0905403106
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发表时间：
2009-08-25
影响因子：
11.1
通讯作者：
Karbstein, Katrin
中科院分区：
文献类型：
--
作者：
Lamanna, Allison C.;Karbstein, Katrin
核糖体组装是一个层次化的过程，涉及核糖体蛋白的前rRNA折叠、修饰以及切割和组装。在真核生物中，这一过程需要一个由200多个蛋白质和RNA组成的大分子复合体。虽然rRNA修饰机制已经有了很好的描述，但对rRNA裂解释放成熟rRNA的了解很少，在酵母中，只有2种内切酶被鉴定出来。必需和保守的核糖体组装因子NOB1被认为是负责通过D位裂解产生18SRRNA成熟的3‘端的内切酶。我们提供了证据，重组NOB1形成了一个四聚体，直接与含有裂解位点D的前rRNA类似物结合。对NOB1’S对一系列RNA截断的亲和力以及对前rRNA的体内外依赖保护的分析表明，NOB1‘S结合位点集中在18SRRNA的3’端，我们的数据也定位于NOB1‘S建议的活性位点。因此，Nob1在18S rRNA的3‘端有切割的准备。结合先前的数据，这些结果强烈表明Nob1参与了D位点的切割。此外，我们的数据提供了证据，表明18S rRNA 3‘端的切割位点是单链的，而不是通常所描述的双链的一部分。利用这些结果，我们建立了NOB1‘S与前玻色体相互作用的模型。
Ribosome assembly is a hierarchical process that involves pre-rRNA folding, modification, and cleavage and assembly of ribosomal proteins. In eukaryotes, this process requires a macromolecular complex comprising over 200 proteins and RNAs. Whereas the rRNA modification machinery is well-characterized, rRNA cleavage to release mature rRNAs is poorly understood, and in yeast, only 2 of 8 endonucleases have been identified. The essential and conserved ribosome assembly factor Nob1 has been suggested to be the endonuclease responsible for generating the mature 3'-end of 18S rRNA by cleaving at site D. Here we provide evidence that recombinant Nob1 forms a tetramer that binds directly to pre-rRNA analogs containing cleavage site D. Analysis of Nob1's affinity to a series of RNA truncations, as well as Nob1-dependent protections of pre-rRNA in vitro and in vivo demonstrate that Nob1's binding site centers around the 3'-end of 18S rRNA, where our data also locate Nob1's suggested active site. Thus, Nob1 is poised for cleavage at the 3'-end of 18S rRNA. Together with prior data, these results strongly implicate Nob1 in cleavage at site D. In addition, our data provide evidence that the cleavage site at the 3'-end of 18S rRNA is single-stranded and not part of a duplex as commonly depicted. Using these results, we have built a model for Nob1's interaction with preribosomes.