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The Molecular Basis of Liquid-like Structure of the Nucleolus

核仁液体状结构的分子基础

基本信息

批准号：
9696544
负责人：
RICHARD W KRIWACKI
金额：
$ 7.8万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-08-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9696544
关键词：
Abbreviations Address Amino Acid Sequence Antibodies Area Binding Biogenesis Birth C-terminal Cancer Cell Growth Cell Nucleolus Cells Cellular Assay Cytoplasmic Granules Development Disease Exhibits Fluorescence Fluorescence Recovery After Photobleaching Fluorescence Resonance Energy Transfer Future Human In Vitro Knowledge Laboratories Length Libraries Liquid substance Location Mediating Membrane Molecular N-terminal NPM1 gene Neutrons Normal Cell Nuclear Nuclear Magnetic Resonance Nucleic Acid Binding 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 single molecule structural biology

项目摘要

SUMMARY 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.

总结这种应用解决了核仁的液体样特征的分子基础，介导核糖体生物发生和某些类型的应激信号传导的核细胞器（例如，包括p53）。核仁具有三个结构区域，纤维中心（FC），致密纤维组分（DFC）和颗粒组分（GC），是核糖体RNA（rRNA）和蛋白质中各个步骤的位置在核糖体生物发生过程中的加工和组装。最近，Brangwynne等人，结果表明，核仁表现出类似于其他点状无膜细胞器的液体样特征。此外，Brangwynne先前表明，点状P颗粒通过以下方式形成液体状结构：其组分的相分离。最近，罗森证明，多价结合相互作用蛋白质中的结构域和基序与相分离有关，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，具有液体状结构特征的无膜细胞器的形成。多功能磷蛋白，核磷蛋白1（NPM 1;在此称为“Npm”），是的GC的核仁，我们假设是一个主要的驱动程序的相分离，产生了 GC的液体状特征。我们最近描述了人的五聚体N-末端结构域的结构， Npm（N130）及其与已知核仁结合配偶体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，一个在其五聚体结构内（称为“A1”），另一个在其五聚体结构内（称为“A2”）。在10个残基长的无序C-末端区段（称为“A2”）内;在N130五聚体内，A1和A2 创造多价性。我们还发现，许多Npm的核仁结合伴侣表现出无序的含有多个含Arg残基基的基序（称为“R基序”）的区域; 结合配偶体也表现出多价性。在目前未发表的研究中，我们已经表明，N130形式在与衍生自Npm配偶体的各种含R基序的肽结合后形成液体样液滴。NPM也在其C-末端显示折叠的核酸结合结构域，提供额外水平的多价性，与核仁中的rRNA相互作用。我们假设Npm瞬时和混杂地与蛋白质和核糖体RNA在核仁中，成核其液体样特征和组织分子功能驱动核糖体的生物合成。