Stepwise assembly of the earliest precursors of large ribosomal subunits in yeast.
Stepwise assembly of the earliest precursors of large ribosomal subunits in yeast.
复制标题
酵母中大核糖体亚基最早前体的逐步组装
DOI：
10.1093/nar/gkx254
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发表时间：
2017-06-20
影响因子：
14.9
通讯作者：
Ye K
中科院分区：
文献类型：
--
作者：
Chen W;Xie Z;Yang F;Ye K
摘要 在酿酒酵母中，小核糖体亚基共转录组装在新生的前体 rRNA 上。目前尚不清楚 60S 大核糖体亚基的高度缠绕结构最初是如何形成的。在这里，我们亲和纯化并分析了一系列在体内组装在长度不断增加的质粒编码的前rRNA片段上的前60S颗粒，揭示了34个反式作用组装因子（AF）、30个核糖体蛋白和5S rRNA的时空组装图。 AF 和核糖体蛋白与 pre-rRNA 片段的逐渐关联有力地支持了 pre-60S 是共转录组装的，而不是转录后组装的。 pre-rRNA 中的内部和外部转录间隔区 ITS1、ITS2 和 3΄ ETS 必须在 pre-60S 中进行加工。我们表明，ITS1 和 ITS2 的加工机制主要分别由前 27S RNA 的 5΄ 和 3΄ 半部分招募。然而，ITS1和ITS2的处理都需要完整的25S区域。 3΄ ETS 在核糖体组装中起次要作用，但对于有效的 rRNA 加工和核糖体成熟很重要。我们还确定了 ITS2 处理之前发生的独特的 60 年代前状态。我们的数据揭示了大核糖体亚基难以捉摸的共转录组装途径。
Abstract Small ribosomal subunits are co-transcriptionally assembled on the nascent precursor rRNA in Saccharomyces cerevisiae. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is initially formed. Here, we affinity purified and analyzed a series of pre-60S particles assembled in vivo on plasmid-encoded pre-rRNA fragments of increasing lengths, revealing a spatiotemporal assembly map for 34 trans-acting assembly factors (AFs), 30 ribosomal proteins and 5S rRNA. The gradual association of AFs and ribosomal proteins with the pre-rRNA fragments strongly supports that the pre-60S is co-transcriptionally, rather than post-transcriptionally, assembled. The internal and external transcribed spacers ITS1, ITS2 and 3΄ ETS in pre-rRNA must be processed in pre-60S. We show that the processing machineries for ITS1 and ITS2 are primarily recruited by the 5΄ and 3΄ halves of pre-27S RNA, respectively. Nevertheless, processing of both ITS1 and ITS2 requires a complete 25S region. The 3΄ ETS plays a minor role in ribosome assembly, but is important for efficient rRNA processing and ribosome maturation. We also identified a distinct pre-60S state occurring before ITS2 processing. Our data reveal the elusive co-transcriptional assembly pathway of large ribosomal subunit.