The Functions of the DEAD-Box RNA Helicase Has1 in 60S Ribosome Biogenesis

描述(申请人提供)：核糖体是普遍存在的细胞纳米机器，能够解码信使核糖核酸和催化蛋白质合成。功能性核糖体的形成需要几个前rRNA加工步骤，加上前rRNA的折叠和核糖体蛋白与rRNA的结合。在人类中，这些过程的失调会导致与正常细胞生长和增殖改变相关的疾病，包括癌症。近年来，该领域的焦点一直是确定参与核糖体组装的因素以及它们发挥作用的具体步骤。然而，这些蛋白质的确切作用在很大程度上还没有被研究过。这些因子包括外切酶和内切酶、GTP酶和ATPase、RNA解旋酶以及RNA结合和支架蛋白。RNA解旋酶驱动RNA-RNA、RNA-蛋白质和蛋白质-蛋白质重排，参与RNA代谢的所有过程，包括转录、剪接和翻译。在酿酒酵母中，超过一半的已知RNA解旋酶在核糖体生物发生中发挥作用，这与这一过程是高度调控和定时的一致。该项目的长期目标是研究死盒RNA解旋酶Has1在60S核糖体组装中的功能。利用酿酒酵母，将采用遗传学、分子生物学、生化和蛋白质组学相结合的方法，在60S核糖体组装途径的两个步骤中解决Has1功能的特定问题。Has1什么时候进入和退出前博色体？招募到前乳体依赖于哪些因素？当Has1耗尽时，哪些核糖体蛋白不能稳定地与前乳糖体结合？当Has1耗尽时，哪些组装因子不能进入或退出前体？Has1的蛋白靶点和辅因子是什么？Has1的RNA靶标是什么？Has1在哪些本地RNA环境中起作用？对这些问题的回答将有助于理解该蛋白在核糖体生物发生中的确切功能，并将有助于揭示RNA解旋酶在RNA代谢过程中的更具体作用。此外，由于核糖体组装的途径在真核生物中高度保守，拟议的研究计划将提供有关这一过程的失调如何导致人类疾病的洞察。 公共卫生相关性：Has1解旋酶和核糖体生物发生途径在真核生物中都高度保守。此外，人类Has1的同源物对细胞生长很重要，它的抑制已被证明可以阻止肿瘤细胞的增殖。因此，了解该解旋酶在酵母中的功能将对进一步研究人类核糖体的生物发生和疾病具有重要意义。

DESCRIPTION (provided by applicant): Ribosomes are ubiquitous cellular nanomachines that enable decoding of mRNA and catalyze protein synthesis. Formation of functional ribosomes requires several pre-rRNA processing steps coupled with folding of the pre-rRNA and binding of ribosomal proteins to the rRNA. In humans, dysregulation of these processes leads to diseases related to alterations in normal cell growth and proliferation, including cancer. In recent years, the focus of the field has been to identify the factors involved in ribosome assembly and the specific steps in which they function. However, the exact roles of these proteins have been largely unstudied. These factors include exo- and endonucleases, GTP- and ATPases, RNA helicases, and RNA binding and scaffolding proteins. RNA helicases drive RNA-RNA, RNA-protein, and protein-protein rearrangements and are involved in all processes of RNA metabolism including transcription, splicing, and translation. In the yeast Saccharomyces cerevisiae, over half of the known RNA helicases function in ribosome biogenesis, consistent with this process being highly regulated and timed. The long-term goal of this project is to investigate the functions of the DEAD- box RNA helicase Has1 in 60S ribosome assembly. Using S. cerevisiae, combined genetic, molecular biological, biochemical, and proteomic approaches wil be carried out to address specific questions of Has1 function in two steps of the 60S ribosome assembly pathway. When does Has1 enter and exit preribosomes? Upon which factors does Has1 depend for recruitment into preribosomes? Which ribosomal proteins fail to stably associate with preribosomes when Has1 is depleted? Which assembly factors fail to enter or exit preribosomes when Has1 is depleted? What are the protein targets and cofactors of Has1? What are the RNA targets of Has1? In what local RNA environments does Has1 function? Answers to these questions will be valuable in understanding the exact functions of this protein in ribosome biogenesis and will shed light on more specific roles of RNA helicases in processes of RNA metabolism. Furthermore, because the pathway of ribosome assembly is highly conserved across eukaryotes, the proposed research plan will provide insight into how dysregulation of this process leads to human disease. PUBLIC HEALTH RELEVANCE: Both the Has1 helicase and the pathway of ribosome biogenesis are highly conserved across eukaryotes. Furthermore, the human homolog of Has1 is important for cell growth and its inhibition has been shown to prevent tumor cell proliferation. Therefore, understanding the functions of this helicase in yeast will have important implications in further study of human ribosome biogenesis and disease.!