YEAST RIBOSOME BIOGENESIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。我们使用酵母酿酒酵母，以促进遗传，分子生物学，生化和 蛋白质组学方法。核糖体组装始于核仁，rRNA在核仁中转录，与核糖体蛋白结合，经过修饰和初始处理，开始形成成熟的核糖体RNPs。胸前颗粒成熟的后续步骤发生在它们释放时 从核仁到核质，并在它们输出到细胞质时。这种组装途径需要一系列动态的重塑步骤，在这些步骤中，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。在人类中，这一途径的失调会导致许多与细胞生长或增殖改变相关的疾病，包括癌症。 筛选核糖体生物发生缺陷的酵母突变株，开发纯化核糖体组装中间体并鉴定其成分的方法，导致鉴定&gt；170反式作用 核糖体组装所需的因子。要了解核糖体生物发生的机制，关键是要弄清楚这些组装因子中的每一个所扮演的确切角色。他们中的哪一个接触了前rRNA？哪些蛋白质相互作用？人们能像在体外组装的原核糖体那样，在新生的rRNPs中定义组装邻域吗？这些因子以什么顺序与前乳糖体相联系，执行它们的功能，然后从粒子中解离？组装因子和核糖体蛋白通过什么方式被招募到前核糖体，被激活，然后从前rRNPs中释放出来？实验就是为了解决这些问题而提出的。 我们专注于60S核糖体亚基前体成熟的两个特定连续步骤--66SA3组装中间体的成熟，随后是66Sb颗粒的成熟。为了开始建立因子作用机制的范式，我们将解决上述问题 装配中这两个步骤所需的几个因素。此外，我们还将更详细地研究Dead-box蛋白Drs1的作用。这个假定的ATPase是否通过触发与前rRNA结合的负调控因子的释放而使66Sb颗粒成熟，并防止其过早切割？ 真核核糖体生物发生所涉及的分子的高度保守性保证了在酵母中发现的核糖体生物发生的原理将为研究更多的多样性提供蓝图 后生动物中核糖体组装的调节方式，包括在许多疾病中被破坏的方式。

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.