The role of nucleolar enzymes in yeast ribosome biogenesis

核糖体是所有生物体内合成蛋白质的细胞器。它们是由两个亚基（一个大亚基和一个小亚基）结合而成的，这两个亚基由大RNA分子和多种蛋白质组成。核糖体的关键成分是核糖体rna (RNAs)，因为它们直接参与蛋白质合成的不同步骤。核糖体的生物发生是一个重要的细胞过程，是细胞生长和增殖的先决条件。在真核生物中，这个过程发生在核仁中，核仁是细胞核的一个突出的隔室。大多数核仁因子实际上在导致成熟rna产生的复杂途径的各个步骤中起作用。我的研究项目主要集中在酵母的核糖体生物发生，酿酒酵母是该领域研究最好的真核生物模型系统。我实验室的工作有助于确定参与成熟rnas产生的核仁因子的分子机制。特别地，我们专注于从前体rRNA分子中去除额外序列的裂解反应。我的实验室正在使用酵母作为模型系统，以利用其具有良好特征的遗传，生化和分子生物学工具。我们未来五年的目标是确定三种核仁酶：Dbp4， Kre33和Nop38的作用。Dbp4是一种RNA解旋酶，在rrna前裂解过程中作为分子马达重新排列RNA-RNA（或可能是RNA-蛋白）相互作用。重要的是，Dbp4与其他核仁因子相关，这些因子在物种中是保守的：它们都是生长所必需的，表明它们执行关键的细胞功能。这些蛋白的人类同源物参与胚胎发育和癌症（Dbp4的人类同源物DDX10已被确定为一种“癌症基因”）。Kre33是一种假定的RNA乙酰转移酶，从细菌到人类是保守的。这种高度的保守性表明它具有关键的细胞功能，但对Kre33在酵母中的作用几乎一无所知。我的实验室发现了Kre33的特定特征，这些特征将它与细菌祖先区分开来，我们怀疑这些特征是Kre33在酵母和人类中功能的关键因素。为了确定Kre33在分子水平上的功能，我们将结合遗传和生化方法。这些研究对于理解为什么Kre33对细胞存活至关重要至关重要。Nop38是一种假定的RNA甲基转移酶，高度保守，但不如Kre33保守。Nop38存在于酵母、植物和动物中，但并不是在所有细菌中都存在，只有那些被称为“极端微生物”的细菌才存在，它们生活在非常恶劣的环境中，比如海洋热喷口；“正常”细菌，比如那些生活在动物内脏里的细菌，没有Nop38。我们对研究这种酶感到非常兴奋，因为我们对它的功能一无所知，一切都有待发现。我们对Dbp4， Kre33和Nop38的研究对于了解这些蛋白在细胞中的功能非常重要：阐明真核生物核糖体生物发生的分子机制可以为改善植物生长，治疗疾病或靶向影响人类和其他动物的真核病原体开辟新的途径。

Ribosomes are the organelles that synthesize proteins in all living organisms. They are formed by the association of two subunits (a small and a large) that are constituted of large RNA molecules and multiple proteins. The key components of ribosomes are the ribosomal RNAs (rRNAs) as they are directly involved in the different steps of protein synthesis. The biogenesis of ribosomes is a major cellular process, and it is a prerequisite for cells to grow in size and proliferate. In eukaryotes, this process takes place in the nucleolus, a prominent compartment of the cell nucleus. The majority of nucleolar factors actually function at various steps of the intricate pathway that leads to the production of mature rRNAs. My research program focuses on ribosome biogenesis in the yeast Saccharomyces cerevisiae, the best studied eukaryotic model system in the field. Work in my laboratory contributes to defining the molecular mechanisms involving nucleolar factors that participate in the production of mature rRNAs. In particular, we focus on the cleavage reactions that remove extra sequences from precursor rRNA molecules. My lab is using yeast as a model system to take advantage of its well-characterized genetic, biochemical and molecular biology tools. Our objective for the next five years is to define the role of three nucleolar enzymes: Dbp4, Kre33 and Nop38. Dbp4 is an RNA helicase that acts as a molecular motor to rearrange RNA-RNA (or possibly RNA-protein) interactions during the pre-rRNA cleavage steps. Importantly, Dbp4 is associated with other nucleolar factors that are conserved among species: all are essential for growth, indicating that they perform crucial cellular functions. Human homologues of these proteins are involved in embryonic development and cancer (the human homologue of Dbp4, DDX10, has been identified as a "cancer gene"). Kre33 is a putative RNA acetyltransferase that is conserved from bacteria to humans. This high degree of conservation suggests it has a critical cellular function but almost nothing is known about the role of Kre33 in yeast. My lab identified specific features of Kre33 that distinguish it from its bacterial ancestors, and we suspect these are key elements for the function of Kre33 in yeast and humans. To determine how Kre33 functions at the molecular level we will combine genetic and biochemical approaches. These investigations are fundamental to understand why Kre33 is essential for cell survival. Nop38 is a putative RNA methyltransferase that is highly conserved but not as much as Kre33. Nop38 is present in yeasts, plants, animals but it is not found in all bacteria, only those termed “extremophiles”, which live in very hostile environments such as oceanic thermal vents; “normal” bacteria, like those that live in the guts of animals, do not have Nop38. We are very excited to work on this enzyme because nothing is known about its function, and everything remains to be discovered. Our research on Dbp4, Kre33 and Nop38 is important to understand how these proteins function in the cell: elucidation of molecular mechanisms underlying ribosome biogenesis in eukaryotes could open new avenues to improve the growth of plants, to treat diseases or to target eukaryotic pathogens that affect humans and other animals.