Identifying Critical Interactions in the Unique Trypanosoma brucei 5S Ribonucleoprotein Complex and their Role in Ribosome Biogenesis

摘要 布鲁氏锥虫是引起人类非洲锥虫病(HAT)的一种真核寄生虫， 那里有7000万高危人口。如果不治疗，HAT几乎总是致命的，但目前 药物价格昂贵，难以管理，而且充满了副作用。因此，有必要 用于开发新药。其中一种方法是将进程作为目标，这些进程对 寄生虫的存活和病原体的特异性。核糖体生物发生是组装大量蛋白质的过程 和核糖体RNA组分进入成熟的功能核糖体。核糖体生物发生的一个关键过程 是5S核糖核蛋白(RNP)复合体的形成和并入到发育中的60S核糖体 亚单位。最近的高分辨率冷冻电子显微镜结构已经确定了关键的亚基间和亚基内 5S RNP复合体成员与60S亚基之间的相互作用，导致构象移动 在核糖体的蛋白质和RNA成分中。因此，纳入5S RNP起着至关重要的作用 调节检查点，这一过程的中断导致核糖体成熟在 60S亚基。鉴于功能核糖体是生存所必需的，这使得组装和 5S RNP复合体的纳入是未来药物开发的一个有希望的目标。我们实验室的工作已经完成 鉴定出锥虫特异性蛋白P34/P37是布鲁氏锥虫5S RNP的一个独特而重要的部分。 我们还展示了P34/P37与蛋白质L5和5S rRNA之间的直接和独特的体外相互作用， 5S RNP的两个经过充分研究的组件。最近，我们鉴定了布鲁氏毛滴虫L11蛋白的同源物， Rpf2和Rrs1，参与5S RNP在酵母中的成熟和掺入。我们假设 这些同源物形成了同源和独特成分之间的相互作用网络 5S RNP复合体，对其形成和整合至关重要。具体目标将是 该项目的重点如下： 1)研究布鲁氏锥虫L11、Rpf2/Rrs1同系物与其他 5S RNP复合体的同源和独特成分的体外研究。 2)利用RNAi敲打技术确定L11和Rpf2/Rrs1在核糖体生物发生中的体内重要性。 沿着细胞线走下去。 在许多撒哈拉以南非洲国家，这仍然是人口的重大疾病负担，部分原因是 与目前的治疗方法相关的许多问题。利用小分子靶向独特的蛋白质 P34/P37及其作为5S RNP复合体成员的重要作用是开发新的 对抗帽子的治疗方法。因此，描述两国之间相互作用的网络 布氏锥虫5S RNP复合体的成员将为小卫星的发展开辟未来道路 分子化疗药物作为治疗HAT的新方法。

ABSTRACT Trypanosoma brucei is a eukaryotic parasite responsible for the disease Human African Trypanosomiasis (HAT), which has an at-risk population of 70 million. HAT is nearly always fatal without treatment, but current medications are expensive, difficult to administer, and rife with adverse side effects. Therefore, there is a need for the development of new drugs. One approach to which is targeting processes that are both essential for parasite survival and pathogen-specific. Ribosome biogenesis is the process of assembling numerous protein and ribosomal RNA components into mature, functional ribosomes. One critical process of ribosome biogenesis is the formation and incorporation of the 5S ribonucleoprotein (RNP) complex into developing 60S ribosomal subunits. Recent high resolution cryo-electron microscopy structures have identified key inter- and intra-subunit interactions between members of the 5S RNP complex and the 60S subunit, which lead to conformational shifts in protein and RNA components of the ribosome. Therefore, the incorporation of the 5S RNP acts as a crucial regulatory checkpoint, with disruption of the process resulting in cessation of ribosome maturation at the level of the 60S subunit. Given that functional ribosomes are required for survival, this makes the assembly and incorporation of the 5S RNP complex a promising target for future drug development. Work in our laboratory has identified the trypanosome-specific proteins P34/P37 as a unique and essential part of the T. brucei 5S RNP. We have also shown direct and unique in vitro interactions between P34/P37 and the protein L5 and 5S rRNA, two well-studied components of the 5S RNP. Recently, we identified T. brucei homologues of the proteins L11, Rpf2 and Rrs1, which are involved in maturation and incorporation of the 5S RNP in yeast. We hypothesize that these homologues form a network of interactions between homologous and unique components of the 5S RNP complex, and are crucial for its formation and incorporation. The specific aims that will be the focus of this project are the following: 1) To characterize the interactions between the T. brucei homologues of L11, Rpf2/Rrs1 and other homologous and unique components of the 5S RNP complex using in vitro studies. 2) Determine the in vivo importance of L11 and Rpf2/Rrs1 in ribosome biogenesis using RNAi knock- down cell lines. HAT remains a significant disease burden on populations in many sub-Saharan African countries, in part due to the many problems associated with current treatments. Using small molecules to target the unique proteins P34/P37 and their essential role as members of the 5S RNP complex is a potential path to developing new treatments to combat HAT. Therefore, characterizing the network of interactions occurring between members of the 5S RNP complex in T. brucei will set a future path toward the development of small molecule chemotherapeutics as novel treatments for HAT.