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: 10318994
• 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/10318994
• 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; West Nile virus; Yeasts; biological adaptation to stress; cancer type; cofactor; combinatorial; crosslink; density; design; developmental disease; experimental study; genetic manipulation; helicase; human disease; in vivo; innovation; milligram; mutant; novel; particle; protein complex; protein function; reconstitution; reconstruction; 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）复合物的组装对于诸如蛋白质的过程至关重要 翻译，mRNA剪接和端粒维护。 Dead-Box家族的ATPases是 无处不在，高度保守的酶，在RNP组装过程中起着重要作用 生活的王国。死盒蛋白在人类健康和疾病中起关键作用；缺陷 死盒蛋白是特定癌症和发育的发展的基础 疾病，由HIV和West Nile等RNA病毒选择，用于病毒RNA加工。 尽管它们是SF2旋转酶家族的一部分，但死盒蛋白缺乏关键领域 存在于过程中SF2解旋酶中，而依靠调节ATP的反式因子 水解和底物结合。在RNP组装过程中，已经提出了ATPase活性 驱动次级和第三级RNA结构的重塑，协调有序的 添加蛋白质以形成功能性RNP组件。反式调节器的要求 使死盒Atpases理想调节剂，将RNP生物发生与细胞信号传导相结合。 但是，由于它们相互作用的瞬时性质，我们没有分子理解 Dead-Box蛋白如何参与和重塑其RNP组件底物。这 提案描述了一种定义四个必需死盒的分子细节的混合方法 蛋白质（dbp10，drs1，spb4和mak5）在复杂的RNP组装过程中，大型 核糖体（60s）亚基。我们通常操纵酵母菌菌株以捕获并丰富不同的酵母菌菌株 瞬态死箱·RNP中间体。这些动态的结构表征 通过冷冻电子显微镜进行复合物，作为综合方法的一部分，包括跨 将质量光谱法连接并针对体外重构实验，将揭示 死盒蛋白与底物RNA的分子相互作用并调节共同 因素。因为60年代成熟的死箱调制与 调节核60年代之前的释放，我们将使用颜色切换的酵母菌菌株来探测 影响DBP10，DRS1，MAK5和SPB4捕获突变体的表达在亚细胞上 60s中间体的分布。这些研究共同代表了一种独特的方法 了解中心重要的60年代生物发生中死盒蛋白的功能 路径。这些创新的试剂及其在集成的实验中的使用 方法将独特地告知死盒蛋白如何参与瞬态，动态 中间体调节RNP组件。