Biogenesis of the Small Ribosomal Subunit in Eukaryotes

描述(由申请人提供)： 所有细胞都需要核糖体进行翻译，这是解码信使RNA和合成蛋白质的过程。为了满足快速分裂的真核细胞的翻译需求，每分钟必须合成数千个核糖体，这个过程消耗了细胞能量预算的很大一部分。事实上，许多癌症的快速发展需要上调核糖体的生物发生。因此，了解调控核糖体生物发生的机制将为开发控制疾病状态下细胞增殖的新工具提供洞察力。核糖体生物发生等基本细胞途径的描绘对于建立翻译研究的智力基础也至关重要。真核核糖体生物发生的许多基本步骤还知之甚少。我们的功能遗传学方法(约翰逊实验室)与体外生化模型的发展(科瑞尔实验室)相结合，使我们在研究真核生物的基本步骤方面处于特别有利的地位。 核糖体生物发生；从核糖体前颗粒(90年代)到40年代以前的颗粒(小亚单位前体)的过渡。我们的总体模型是，甲基转移酶Bud23监控40S组装的状态，只有在RNA完成转录和关键折叠后，才会触发RNA解旋酶Ecm16促进90年代前40秒的释放。我们初步结果的一项主要成就是我们能够捕获ECM16-中间粒子。为了开始解决这一模型，我们的研究检验了四个特定的假设：(I)Ecm16是将U3 snoRNA及其相关蛋白从Pre-rRNA中分离出来的解旋酶；(Ii)Bud23激活了这一解旋酶活性；(Iii)Imp4稳定了ECm16的靶向双链；以及(Iv)核糖体蛋白Rps2伴侣形成中央伪结，这是18S rRNA的一个关键结构特征，其形成受到空间上的阻止，直到Ecm16中断了U3-Pre-rRNA的相互作用。酿酒酵母中的前rRNA加工途径和待研究的基因在高等真核生物中具有同源性。因此，酿酒酵母是我们选择的模式生物，因为它允许我们结合强大的分子遗传学和生化方法。

DESCRIPTION (provided by applicant): All cells require ribosomes for translation, the process of decoding messenger RNA and synthesizing proteins. To satisfy the translational needs of a rapidly dividing eukaryotic cell, thousands of ribosomes must be synthesized per minute in a process that consumes a large portion of the cell's energy budget. Indeed, the rapid growth of many cancers requires up-regulation of ribosome biogenesis. Thus, understanding the mechanisms regulating ribosome biogenesis will provide insight for the development of new tools for controlling cell proliferationin disease states. The delineation of fundamental cellular pathways such as ribosome biogenesis is also critical for building the intellectual foundation for translational research. Many of the fundamental steps of eukaryotic ribosome biogenesis are poorly understood. Our functional genetic approach (Johnson lab) combined with the development of in vitro biochemical models (Correll lab) places us in a particularly strong position to study a fundamental step in eukaryotic ribosome biogenesis; the transition from the pre-ribosome particle (90S) to the pre-40S particle (small subunit precursor). Our overarching model is that the methyltransferase Bud23 monitors that status of 40S assembly, triggering the RNA helicase Ecm16 to promote release of the pre-40S from 90S only after completion of transcription and critical folding of the RNA. A major achievement in our preliminary results is our ability to trap an Ecm16-intermediate particle. To begin to address this model, our studies test four specific hypotheses: (i) Ecm16 is the helicase that dissociates U3 snoRNA and its associated proteins from the pre-rRNA (ii) Bud23 activates this helicase activity; (iii) Imp4 stabilizes the duplex that is the target of Ecm16; and (iv) the ribosomal protein Rps2 chaperones formation of the central pseudoknot, a key structural feature in 18S rRNA whose formation is sterically blocked until Ecm16 disrupts the U3-pre-rRNA interactions. The pre-rRNA processing pathways and the genes to be studied in the yeast Saccharomyces cerevisiae have counterparts in higher eukaryotes. Hence, S. cerevisiae is our model organism of choice because it allows us to combine powerful molecular genetic and biochemical approaches.