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）复合物的组装是蛋白质合成等过程的核心， 翻译、mRNA剪接和端粒维持。死亡盒家族的ATP酶是 普遍存在的，高度保守的酶，在RNP组装过程中发挥重要作用，在所有 生命的王国死亡盒蛋白在人类健康和疾病中起着关键作用; 死亡盒蛋白是特定癌症进展以及发育的基础。 这些蛋白质被RNA病毒如HIV和西尼罗河病毒吸收用于病毒RNA加工。 虽然它们是解旋酶SF 2家族的一部分，但DEAD-盒蛋白缺乏关键结构域 存在于进行性SF 2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，已经提出ATP酶活性 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。转换器的要求 使死亡盒ATP酶成为理想的调节剂，将RNP生物合成与细胞信号传导相结合。 然而，由于它们相互作用的瞬时性质，我们没有分子理解 死亡盒蛋白如何参与并重塑其RNP组装底物。这 一项提案描述了一种混合方法来定义四个基本的死亡盒的分子细节 蛋白质（Dbp 10，Drs 1，Spb 4和Mak 5）在组装一个复杂的RNP，大 核糖体（60 S）亚基。我们对酵母菌株进行了基因改造， 瞬时死亡盒·RNP中间体。这些动态的结构特征 复合物的低温电子显微镜，作为一个综合方法的一部分，包括交叉， 将质谱和体外重建实验相结合， DEAD盒蛋白与底物RNA的分子相互作用以及调节共- 因素由于DEAD盒对60 S成熟的调节与 调节核仁前60 S释放，我们将使用颜色转换酵母菌株来探测 Dbp 10、Drs 1、Mak 5和Spb 4诱捕突变体的表达对亚细胞 60年代的中间人。总之，这些研究代表了一种独特的方法， 了解死亡盒蛋白在重要的60年代生物发生中的功能 通路这些创新试剂及其在综合实验中的应用 这种方法将独特地告知死亡盒蛋白质如何参与瞬时，动态 调节RNP组装的中间体。