Formation and maturation of the eukaryotic 60S ribosomal subunit

核糖体生物发生包括rRNA的转录、修饰、折叠和加工，并结合80个核糖体蛋白形成小(40S)和大(60S)亚基。通过结合细胞生物学、生化和结构方法，我们深入了解了酿酒酵母核糖体形成过程中的各个步骤。在接下来的资助期间，我们将继续对这一最复杂的成熟途径进行结构和功能研究。第一个焦点将是早期核仁60S前颗粒以及属于Brix结构域家族的几个关键蛋白(SSF1、Brx1、Rpf1)在启动60S组装中的作用。在第二个项目中，我们将研究5S RNP组装到60年代前的核糖体中，以及结构成熟是如何发生的，重点是Rpf2-Rrs1复合体和一种新的组装因子Cgr1。在第三个项目中，我们想要分析在60S生物发生过程中，发生肽形成的60S亚单位的催化中心(肽转移酶中心或PTC)是如何形成的，这也涉及从该区域去除因子。具体地说，我们将集中在拆除AAA ATPase Drg1，以及显性负drg1突变是否以及如何影响前核糖体Nog1和Nsa2的释放。最后，在研究Rpl4掺入60年代前的核糖体时，我们发现了一个特定的rpl4突变，它排列在出口隧道内，导致核糖体成熟受阻，推测是在细胞质中。因此，我们想要澄清这种感知出口隧道完整性的质量控制的分子细节。从所有这些计划中的研究中，我们期待对60S亚单位生物发生的机械性洞察，这也可能影响我们对核糖体相关疾病的理解。

Ribosome biogenesis comprises, transcription, modification, folding and processing of the rRNA coupled with the incorporation of 80 ribosomal proteins to form the small (40S) and large (60S) subunit. By combining cell biological, biochemical and structural methods in the model yeast Saccharomyces cerevisiae, we gained insight into various steps during 60S ribosome formation. During the next funding period, we will continue our structural and functional studies on this most complicated maturation pathway. The first focus will be on early nucleolar pre-60S particles and the role of several essential proteins belonging to the Brix domain family (Ssf1, Brx1, Rpf1) for initiating 60S assembly. In a second project, we will study the assembly of the 5S RNP into the pre-60S ribosome and how structural maturation occurs with the focus on the Rpf2-Rrs1 complex and a novel assembly factor Cgr1. In a third project, we want to analyze how the catalytic center of the 60S subunit, where peptide formation occurs (peptidyl transferase center or PTC), is formed during 60S biogenesis, which also involves factor removal from this region. Specifically, we will focus on the dismantling AAA ATPase Drg1, and whether and how dominant-negative drg1 mutants affect Nog1 and Nsa2 release from the pre-ribosome. Finally, upon studying Rpl4 incorporation into the pre-60S ribosome, we came across a specific rpl4 mutation that lines the exit tunnel, which caused a ribosome maturation block at a very late step, presumably in the cytoplasm. Hence, we would like to clarify the molecular details of this quality control sensing the intactness of the exit tunnel. From all these planned studies, we expect mechanistic insight into 60S subunit biogenesis, which could also impact our understanding of ribosome-associated diseases.