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: 9307879
• 负责人: RICHARD W KRIWACKI
• 金额: $ 47.83万
• 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/9307879
• 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（由申请人提供）：本申请解决了核仁的液体样特征的分子基础，核仁是介导核糖体生物发生和某些类型的应激信号传导（例如，包括p53）。核仁具有纤维中心（FC）、致密纤维组分（DFC）和颗粒组分（GC）三个结构区域，它们是核糖体生物发生过程中核糖体RNA（rRNA）和蛋白质加工和组装的各个步骤的位置。最近，Brangwynne等人，表明，GC的核仁表现出液体样的功能，类似于其他点状，无膜细胞器。此外，Brangwynne先前表明，点状P颗粒通过其组分的相分离形成液体状结构。最近，罗森证明了相互作用蛋白质内的结合结构域和基序的多价性与相分离相关，并且McKnight已经表明多价、低复杂性蛋白质序列经历与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，驱动具有液体状结构特征的无膜细胞器的形成。 多功能磷蛋白，Nucleophosmin 1（NPM 1;这里称为“Npm”），是核仁GC的主要成分，我们假设是相分离的主要驱动因素，导致GC的液体样特征。我们最近描述了 人Npm（N130）的五聚体N-末端结构域及其与已知核仁结合伴侣（包括核糖体蛋白）相互作用的分子基础。N130表现出两个酸性区域，一个在其五聚体结构内（称为“A1”），另一个在10个残基长的无序C-末端片段内（称为“A2”）;在N130五聚体内，A1和A2产生多价。我们还发现，许多Npm的核仁结合伙伴表现出无序的区域，包含多个精氨酸残基的基序（称为“R基序”）;在Npm的结合伙伴的多个R基序也表现出多价性。在目前未发表的研究中，我们已经表明N130在与源自Npm伴侣的各种含R基序的肽结合后形成液体状液滴。Npm在其C-末端还表现出折叠的核酸结合结构域，为与核仁中rRNA的相互作用提供了额外水平的多价。我们假设Npm与核仁中的蛋白质和rRNA短暂且混杂地相互作用，使其液体样特征成核，并组织驱动核糖体生物发生的分子功能。