The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor.
The Npa1p complex chaperones the assembly of the earliest eukaryotic large ribosomal subunit precursor.
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DOI：
10.1371/journal.pgen.1007597
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发表时间：
2018-08
期刊：
影响因子：
4.5
通讯作者：
Henry Y
中科院分区：
文献类型：
--
作者：
Joret C;Capeyrou R;Belhabich-Baumas K;Plisson-Chastang C;Ghandour R;Humbert O;Fribourg S;Leulliot N;Lebaron S;Henras AK;Henry Y
产生大核糖体亚基的早期步骤可能是真核核糖体生物发生中最不为人所知的阶段。酵母大核糖体亚基的第一个特异性前体，即第一个pre-60S颗粒，包含30个组装因子（AFs），包括8个RNA解旋酶。这些解旋酶被认为可以驱动构象重排，但在体外通常缺乏底物特异性。它们在核糖体前颗粒中靶向正确底物的机制及其精确的分子作用在很大程度上仍然未知。我们发现，在啤酒酵母中，对于第一个60s前核糖体颗粒的正常积累至关重要的Dbp6p解旋酶，在进入90S前核糖体颗粒之前，与四种AFs复合物（即Npa1p， Npa2p， Nop8p和Rsa3p）相关。通过串联亲和纯化，我们发现Npa1p形成了该复合物的主链。我们提供的证据表明，Npa1p和Npa2p直接结合Dbp6p，我们证明，Npa1p是必不可少的插入Dbp6p解旋酶在90S核糖体前颗粒。此外，通过体内交联分析（CRAC），我们在25S rRNA的第一和最后二级结构域的根螺旋附近绘制了Npa1p rRNA结合位点。这一发现支持了Npa1p和Dbp6p在大亚基rnas的根螺旋的形成和/或聚集中起作用的观点，这些大亚基rnas形成了大核糖体亚基RNA结构的核心。Npa1p还交联到参与解码中心和肽基转移酶中心修饰的snoRNAs，以及这些snoRNAs在25S rRNA上的结合位点附近。我们的数据表明，Dbp6p解旋酶和Npa1p复合物在25S rRNA核心的压实和snoRNA-pre-rRNA相互作用的控制中起着关键作用。核糖体是合成蛋白质的分子机器，由大小亚基组成，由许多核糖体蛋白（RPs）与适当折叠的核糖体rna （RNAs）结合而成。RP结合以及rrna的加工和折叠发生在一系列核糖体前颗粒中。初始前60s颗粒的形成是核糖体亚基的第一个前体，是真核生物中核糖体生物发生的最不为人所知的步骤。这种核糖体前颗粒含有几种组装因子（AFs），包括被认为催化关键构象重排的RNA解旋酶。这些解旋酶本身通常缺乏底物特异性。在这里，我们发现Dbp6p解旋酶是第一个pre-60S颗粒的一个组成部分，对其正常积累至关重要，它与包括Npa1p在内的四种AFs复合物相关。我们证明Npa1p直接结合Dbp6p，形成复合体的主干，并且是Dbp6p在核糖体前颗粒内整合所必需的。我们发现Npa1p结合形成大亚基RNAs核心的序列，以及化学修饰大亚基RNAs所需的小核仁rna。总之，我们的研究结果表明，Npa1p复合物在大亚基rna的化学修饰和折叠中起着至关重要的作用。
The early steps of the production of the large ribosomal subunit are probably the least understood stages of eukaryotic ribosome biogenesis. The first specific precursor to the yeast large ribosomal subunit, the first pre-60S particle, contains 30 assembly factors (AFs), including 8 RNA helicases. These helicases, presumed to drive conformational rearrangements, usually lack substrate specificity in vitro. The mechanisms by which they are targeted to their correct substrate within pre-ribosomal particles and their precise molecular roles remain largely unknown. We demonstrate that the Dbp6p helicase, essential for the normal accumulation of the first pre-60S pre-ribosomal particle in S. cerevisiae, associates with a complex of four AFs, namely Npa1p, Npa2p, Nop8p and Rsa3p, prior to their incorporation into the 90S pre-ribosomal particles. By tandem affinity purifications using yeast extracts depleted of one component of the complex, we show that Npa1p forms the backbone of the complex. We provide evidence that Npa1p and Npa2p directly bind Dbp6p and we demonstrate that Npa1p is essential for the insertion of the Dbp6p helicase within 90S pre-ribosomal particles. In addition, by an in vivo cross-linking analysis (CRAC), we map Npa1p rRNA binding sites on 25S rRNA adjacent to the root helices of the first and last secondary structure domains of 25S rRNA. This finding supports the notion that Npa1p and Dbp6p function in the formation and/or clustering of root helices of large subunit rRNAs which creates the core of the large ribosomal subunit RNA structure. Npa1p also crosslinks to snoRNAs involved in decoding center and peptidyl transferase center modifications and in the immediate vicinity of the binding sites of these snoRNAs on 25S rRNA. Our data suggest that the Dbp6p helicase and the Npa1p complex play key roles in the compaction of the central core of 25S rRNA and the control of snoRNA-pre-rRNA interactions. Ribosomes, the molecular machines synthesizing proteins, are composed of a small and large subunit, formed by the binding of numerous ribosomal proteins (RPs) to properly folded ribosomal RNAs (rRNAs). RP incorporation as well as processing and folding of rRNAs occur within a succession of pre-ribosomal particles. Formation of the initial pre-60S particle, the first precursor to the large ribosomal subunit, is the least understood step of ribosome biogenesis in eukaryotes. This pre-ribosomal particle contains several assembly factors (AFs), including RNA helicases believed to catalyse key conformational rearrangements. These helicases usually lack substrate specificity on their own. Here, we show that the Dbp6p helicase, a component of the first pre-60S particle and essential for its normal accumulation, associates with a complex of four AFs, including Npa1p. We demonstrate that Npa1p directly binds Dbp6p, forms the backbone of the complex and is required for the integration of Dbp6p within pre-ribosomal particles. We show that Npa1p binds to sequences forming the core of large subunit rRNAs as well as small nucleolar RNAs required for chemical modification of large subunit rRNAs. Altogether our results suggest that the Npa1p complex plays a crucial role in the chemical modification and folding of large subunit rRNAs.