YEAST RIBOSOME BIOGENESIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 这个项目的长期目标是了解核糖体是如何在真核生物中组装的。 我们使用酿酒酵母，以促进基因，分子生物学，生物化学， 蛋白质组学方法 核糖体组装起始于核仁，在核仁中rRNA被转录，与核糖体蛋白质结合，并经历修饰和初始加工以开始形成成熟的核糖体RNP。 前核糖体颗粒成熟的后续步骤发生在它们释放后 从核仁到核质，并在它们出口到细胞质时。 这种组装途径需要一系列动态的重塑步骤，其中蛋白质-蛋白质，RNA-蛋白质和RNA-RNA相互作用被建立，破坏和重新配置。 人类中该途径的失调导致许多与细胞生长或增殖改变相关的疾病，包括癌症。 筛选核糖体生物合成缺陷的酵母突变体，以及纯化核糖体组装中间体和鉴定其组分的方法的发展，导致鉴定了>170个反式作用的 核糖体组装所需的因子。理解核糖体生物发生机制的核心是弄清楚这些组装因子中的每一个所扮演的确切角色。 哪一个会接触前rRNA？ 哪些蛋白质相互作用？人们能否像在体外组装的原核核糖体那样，在新生的rRNP中定义组装邻域？ 这些因子以什么顺序与前核糖体结合，执行它们的功能，然后从颗粒中分离？组装因子和核糖体蛋白通过什么方式被募集到前核糖体，激活，然后从前rRNP释放？ 实验提出了解决这些问题。 我们专注于两个特定的连续步骤，在成熟的前体成熟60 S核糖体亚基-成熟的66 SA 3组装中间体，其次是66 SB颗粒。为了开始建立因子功能机制的范例，我们将解决上述问题， 这两个组装步骤需要几个因素。 此外，我们将更详细地研究死亡盒蛋白Drs 1的作用。这种假定的ATP酶是否通过触发与前rRNA结合并防止其过早裂解的负调节剂的释放而使66 SB颗粒成熟？ 参与真核生物核糖体生物发生的分子的高度保守性预示着在酵母中发现的核糖体生物发生的原则将为研究更多样化的核糖体提供蓝图。 后生动物中核糖体组装的调节模式，包括在许多疾病中被破坏的核糖体组装。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The long term goal of this project is to understand how ribosomes are assembled in eukaryotes. We use the yeast Saccharomyces cerevisiae, to facilitate combined genetic, molecular biological, biochemical, and proteomic approaches. Ribosome assembly initiates in the nucleolus, where rRNA is transcribed, associates with ribosomal proteins, and undergoes modification and initial processing to begin to form mature ribosomal RNPs. Subsequent steps in maturation of preribosomal particles occur upon their release from the nucleolus to the nucleoplasm and upon their export to the cytoplasm. This assembly pathway requires a dynamic series of remodeling steps in which protein-protein, RNA-protein, and RNA-RNA interactions are established, disrupted and reconfigured. Dysregulation of this pathway in humans leads to many diseases related to alterations in cell growth or proliferation, including cancer. Screens for yeast mutants defective in ribosome biogenesis, and development of methods to purify ribosome assembly intermediates and identify their constituents, led to the identification of >170 trans-acting factors required for ribosome assembly. Central to understand the mechanisms of ribosome biogenesis will be to figure out the precise roles played by each of these assembly factors. Which of them contacts pre-rRNA? Which proteins interact with each other? Can one define assembly neighborhoods within the nascent rRNPs, as observed for prokaryotic ribosomes assembled in vitro? In what order do these factors associate with preribosomes, carry out their functions, then dissociate from the particles? By what means are assembly factors and ribosomal proteins recruited to pre-ribosomes, activated, then released from the pre-rRNPs? Experiments are proposed to address these questions. We are focusing on two particular consecutive steps in the maturation of precursors to mature 60S ribosomal subunits--maturation of the 66SA3 assembly intermediates, followed by the 66SB particles. To begin to establish paradigms for mechanisms of factor function, we will address the above questions for several factors required for these two steps in assembly. In addition, we will investigate in more detail the role of the DEAD-box protein Drs1. Does this putative ATPase enable maturation of 66SB particles, by triggering the release of negative regulators that bind to pre-rRNA and prevent its premature cleavage? The high degree of conservation of molecules involved in eukaryotic ribosome biogenesis promises that principles governing ribosome biogenesis discovered in yeast will provide blueprints to study more diverse modes of regulation of ribosome assembly in metazoans,including those disrupted in many diseases.