Integrated studies of DEAD-box ATPase function during large ribosomal subunit maturation

大核糖体亚基成熟过程中 DEAD-box ATP 酶功能的综合研究

• 批准号: 10543093
• 负责人: Jan Peter Erzberger
• 金额: $ 34.02万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543093
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affinity; Binding; Biochemical; Biogenesis; Biological; Biological Models; Biophysics; Cell physiology; Cells; Color; Complex; Coupling; Cryoelectron Microscopy; Cytolysis; Defect; Disease; Dominant-Negative Mutation; Elements; Ensure; Enzymes; Family; Freezing; Genetic; Genetic Recombination; HIV; Health; Heart; Heterogeneity; Hour; Human; Hybrids; In Vitro; Inherited; Kinetics; Life; Light; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Messenger RNA; Methodology; Molecular; Monitor; Movement; Nature; Pathway interactions; Play; Point Mutation; Process; Proteins; Proteome; Protocols documentation; Publishing; Quality Control; RNA; RNA Processing; RNA Splicing; RNA Viruses; Reagent; Regulation; Research; Resolution; Ribonucleoproteins; Ribosomal Biogenesis Pathway; Ribosomes; Role; Sampling; Signal Transduction; Structure; Telomere Maintenance; Testing; Time; Translations; Virus Diseases; Visualization; West Nile; Yeasts; biological adaptation to stress; cancer type; cofactor; combinatorial; crosslink; density; design; developmental disease; experimental study; genetic manipulation; helicase; human disease; improved; in vivo; innovation; milligram; mutant; novel; particle; protein complex; protein function; reconstitution; reconstruction; ribosomopathy; spatiotemporal; tool; tumor progression; viral RNA

The assembly of ribonucleoprotein (RNP) complexes is central to processes such as protein translation, mRNA splicing, and telomere maintenance. ATPases of the DEAD-box family are ubiquitous, highly conserved enzymes that play essential roles during RNP assembly in all kingdoms of life. DEAD-box proteins play critical roles in human health and disease; defects in DEAD-box proteins underlie the progression of specific cancers as well as developmental disorders, and are co-opted by RNA viruses such as HIV and West Nile for viral RNA processing. Though they are part of the SF2 family of helicases, DEAD-box proteins lack key domains present in processive SF2 helicases, and rely instead on trans factors that regulate ATP hydrolysis and substrate binding. During RNP assembly, ATPase activity has been proposed to drive the remodeling of secondary and tertiary RNA structures, coordinating the ordered addition of proteins to form functional RNP assemblies. The requirement of trans modulators makes DEAD-box ATPases ideal regulators, integrating RNP biogenesis with cellular signaling. However, due to the transient nature of their interactions, we have no molecular understanding of the how DEAD-box proteins engage and remodel their RNP assembly substrates. This proposal describes a hybrid approach to define the molecular details of four essential DEAD-box proteins (Dbp10, Drs1, Spb4 and Mak5) during the assembly of a complex RNP, the large ribosomal (60S) subunit. We genetically manipulated yeast strains to trap and enrich distinct, transient DEAD-box·RNP intermediates. The structural characterization of these dynamic complexes by cryo-electron microscopy, as part of an integrated approach that includes cross- linking mass-spectrometry and targeted in vitro reconstitution experiments, will shed light on the molecular interactions of DEAD-box proteins with substrate RNA and modulating co- factors. Because DEAD-box modulation of 60S maturation is closely associated with the regulation of nucleolar pre-60S release, we will use a color-switching yeast strain to probe the effect the expression of Dbp10, Drs1, Mak5 and Spb4 trapping mutants have on the subcellular distribution of 60s intermediates. Together, these studies represent a unique approach to understand the function of DEAD-box proteins in the centrally important 60S biogenesis pathway. These innovative reagents and their use within an integrative experimental approach will uniquely inform how DEAD-box proteins engage transient, dynamic intermediates to modulate RNP assembly.

核糖核蛋白(RNP)复合体的组装是蛋白质等过程的核心 翻译、信使核糖核酸剪接和端粒维持。死亡盒子家族的ATPase是 普遍存在的高度保守的酶，在RNP组装过程中起着至关重要的作用 生命的王国。DEAD-BOX蛋白在人类健康和疾病中发挥关键作用；缺陷 DEAD-BOX蛋白是特定癌症进展和发育的基础 疾病，并被艾滋病毒和西尼罗河等RNA病毒用于病毒RNA加工。 虽然它们是解旋酶SF2家族的一部分，但DEAD-box蛋白缺乏关键结构域 存在于进行性的SF2解旋酶中，并依赖于调节ATP的反式因子 水解和底物结合。在RNP组装过程中，ATPase活性被认为是 推动二级和三级RNA结构的重构，协调有序的 添加蛋白质以形成功能性RNP组件。反式调制器的要求 使死盒ATPase成为理想的调节剂，将RNP生物发生与细胞信号转导相结合。 然而，由于它们相互作用的瞬变性质，我们没有分子上的了解 Dead-box蛋白质如何结合和重塑它们的RNP组装底物。这 提案描述了一种混合方法来定义四个基本死盒的分子细节 蛋白质(Dbp10、Drs1、Spb4和Mak5)在复杂的RNP组装过程中，大的 核糖体(60S)亚单位。我们通过基因操作酵母菌株来捕获和丰富不同的， 暂态死盒·RNP中间体。这些动态结构特征 作为综合方法的一部分，包括交叉电子显微镜。 将质谱学和定向体外重建实验相结合，将有助于揭示 研究了DEAD-box蛋白与底物RNA的分子相互作用及其调控机制。 各种因素。因为60S成熟的死盒调制与 核仁60S前释放的调节，我们将使用一株颜色转换的酵母菌株来探测 Dbp10、Drs1、Mak5和Spb4诱捕突变体对亚细胞表达的影响 60S中间体的分布。总而言之，这些研究代表了一种独特的方法 了解DEAD-box蛋白在中心重要的60S生物发生中的功能 路径。这些创新试剂及其在综合实验中的使用 这种方法将独特地告知死盒蛋白如何参与瞬时、动态的 调节RNP组装的中间体。