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) 复合物的组装对于蛋白质等过程至关重要 DEAD-box 家族的翻译、mRNA 剪接和端粒维持是。 普遍存在、高度保守的酶，在所有 RNP 组装过程中发挥重要作用 死亡盒蛋白在人类健康和疾病中发挥着重要作用； DEAD-box 蛋白是特定癌症进展和发育的基础 疾病，并被 HIV 和西尼罗河等 RNA 病毒选择进行病毒 RNA 加工。 尽管它们是 SF2 解旋酶家族的一部分，但 DEAD-box 蛋白缺乏关键结构域 存在于进行性 SF2 解旋酶中，并依赖于调节 ATP 的反式因子 在 RNP 组装过程中，ATP 酶活性被认为与水解和底物结合有关。 驱动二级和三级RNA结构的重塑，协调有序的 添加蛋白质以形成功能性 RNP 组装体 需要反式调节剂。 使 DEAD-box ATP 酶成为理想的调节剂，将 RNP 生物发生与细胞信号传导相结合。 然而，由于它们相互作用的短暂性，我们没有分子理解 DEAD-box 蛋白如何参与并重塑其 RNP 组装底物。 提案描述了一种混合方法来定义四个基本 DEAD-box 的分子细节 复杂 RNP 组装过程中的蛋白质（Dbp10、Drs1、Spb4 和 Mak5） 我们通过基因操作酵母菌株来捕获和富集不同的核糖体 (60S) 亚基。 这些动态瞬态DEAD-box·RNP中间体的结构表征。 通过冷冻电子显微镜观察复合物，作为综合方法的一部分，包括交叉 将质谱法和靶向体外重组实验联系起来，将揭示 DEAD-box 蛋白与底物 RNA 的分子相互作用并调节共 因为 DEAD-box 调制与 60S 成熟密切相关。 核仁前 60S 释放的调节，我们将使用颜色转换酵母菌株来探测 Dbp10、Drs1、Mak5 和 Spb4 捕获突变体的表达对亚细胞的影响 60 年代中间体的分布，这些研究代表了一种独特的方法。 了解 DEAD-box 蛋白在 60S 生物发生中至关重要的功能 这些创新试剂及其在综合实验中的使用。 该方法将独特地告知 DEAD-box 蛋白如何参与瞬态、动态的 调节 RNP 组装的中间体。