The Molecular Basis of Liquid-like Structure of the Nucleolus

核仁液体状结构的分子基础

• 批准号: 9414888
• 负责人: RICHARD W KRIWACKI
• 金额: $ 1.93万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9414888
• 关键词: Address; Amino Acid Sequence; Area; Binding; Biogenesis; Birth; C-terminal; Cancer Cell Growth; Cell Nucleolus; Cells; Cellular Assay; Cytoplasmic Granules; Development; Disease; Exhibits; Fluorescence; Future; Human; In Vitro; Knowledge; Laboratories; Length; Libraries; Liquid substance; Location; Mediating; Membrane; Molecular; N-terminal; NPM1 gene; Normal Cell; Nuclear; Nucleic Acid Folding; Nucleolar Proteins; Organelles; Peptide Library; Peptides; Phase; Phosphoproteins; Preparation; Property; Protein Biosynthesis; Proteins; RNA; Research Institute; Resolution; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Signal Transduction; Stress; Structure; TP53 gene; Tertiary Protein Structure; Testing; cancer cell; drug discovery; experience; novel therapeutics; nucleophosmin; public health relevance; single molecule; structural biology

 DESCRIPTION (provided by applicant): This application addresses the molecular basis of the liquid-like features of the nucleolus, a membrane-less nuclear organelle that mediates ribosome biogenesis and certain types of stress signaling (e.g., involving p53). The nucleolus exhibits three structural regions, the fibrillar center (FC), dense fibrillar component (DFC), and granular component (GC), that are the locations of various steps in ribosomal RNA (rRNA) and protein processing and assembly during ribosome biogenesis. Recently, Brangwynne, et al., showed that the GC of the nucleolus exhibits liquid-like features, similar to those of other punctate, membrane-less organelles. Furthermore, Brangwynne previously showed that punctate P granules form liquid-like structures through phase separation of their components. More recently, Rosen demonstrated that multi-valency of binding domains and motifs within interacting proteins is associated with phase separation, and McKnight has shown that multi-valent, low complexity protein sequences experience phase separation with RNA. These and other recent studies have given birth to a new area of structural biology: phase separation phenomena that drive formation of membrane-less organelles with liquid-like structural features. The multi-functional phospho-protein, Nucleophosmin 1 (NPM1; termed "Npm" here), is a major constituent of the GC of the nucleolus and we hypothesize is a main driver of the phase separation that gives rise to the GC's liquid-like features. We recently described the structure of the pentameric, N-terminal domain of human Npm (N130) and the molecular basis for its interactions with known nucleolar binding partners, including ribosomal proteins. N130 exhibits two acidic tracts, one within its pentamer structure (termed "A1") and another within a 10 residue-long disordered C-terminal segment (termed "A2"); within the N130 pentamer, A1 and A2 create multi-valency. We additionally showed that many of Npm's nucleolar binding partners exhibit disordered regions containing multiple Arg residue-containing motifs (termed "R motifs"); the multiple R motifs in Npm's binding partners also exhibit multi-valency. In currently unpublished studies, we have shown that N130 forms liquid-like droplets upon binding to various R motif-containing peptides derived from Npm partners. Npm also exhibits a folded nucleic binding domain at its C-terminus, providing an additional level of multi-valency for interactions with rRNA in the nucleolus. We hypothesize that Npm transiently and promiscuously interacts with proteins and rRNA in the nucleolus, nucleating its liquid-like features and organizing the molecular functions that drive ribosome biogenesis.

 描述(申请人提供)：本申请涉及核仁的液样特征的分子基础，核仁是一种无膜的核细胞器，介导核糖体的生物发生和某些类型的胁迫信号(例如，涉及P53)。核仁有三个结构区域，即纤维中心(FC)、致密纤维成分(DFC)和颗粒成分(GC)，它们是核糖体RNA(RRNA)和蛋白质在核糖体生物发生过程中加工和组装的各个步骤的位置。最近，Brangwynne等人发现，核仁的GC表现出类似于其他点状、无膜细胞器的液体状特征。此外，Brangwynne以前曾证明，点状P颗粒通过其组分的相分离形成类液体结构。最近，Rosen证明了相互作用蛋白质中结合结构域和基序的多价性与相分离有关，McKnight证明了多价、低复杂性的蛋白质序列经历了RNA的相分离。这些和其他最近的研究催生了结构生物学的一个新领域：相分离现象，驱动具有类似液体结构特征的无膜细胞器的形成。多功能磷酸蛋白--核磷蛋白1(NPM1；这里称为“NPM”)是核仁GC的主要组成部分，我们假设是导致GC液体样特征的相分离的主要驱动因素。我们最近描述了 人类Npm(N130)的五聚体N-末端结构域及其与已知核仁结合伙伴相互作用的分子基础，包括核糖体蛋白。N130表现出两个酸性区段，一个在其五聚体结构中(称为“A1”)，另一个在10个残基长度的无序C-末端片段(称为“A2”)中；在N130五聚体中，A1和A2产生多价。我们还发现，许多Npm的核仁结合伙伴显示出含有多个Arg残基的无序区域(称为“R基序”)；Npm结合伙伴中的多个R基序也显示出多价。在目前未发表的研究中，我们已经表明N130与来自Npm伙伴的各种含有R基序的多肽结合后形成液滴。NPM还在其C末端显示一个折叠的核结合结构域，为与核仁中的rRNA相互作用提供了额外的多价水平。我们假设Npm与核仁中的蛋白质和rRNA瞬时和混杂地相互作用，使其液状特征成核，并组织驱动核糖体生物发生的分子功能。