Reversible Polymerization of Low Complexity Polypeptide Sequences as a Framework

低复杂度多肽序列的可逆聚合作为框架

• 批准号: 8899608
• 负责人: STEVEN L MCKNIGHT
• 金额: $ 49.09万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899608
• 关键词: Accidents; Acetylation; Adopted; Applications Grants; Binding; Biogenesis; Biological Assay; C-terminal; Cell Extracts; Cell Nucleus; Cells; Chemicals; Complex; Cytoplasmic Granules; DNA Binding Domain; Dimensions; Disease; Eukaryotic Cell; Fiber; Filament; Gel; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Health; Homeobox; Hydrogels; Ice; In Vitro; Incubated; Isoxazoles; Laboratories; Laboratory Chemicals; Lateral; Leucine Zippers; Life; Link; Liquid substance; Mammalian Cell; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Membrane; Messenger RNA; Methods; Microscopic; Molecular; Monitor; Mutation; National Institute of Diabetes and Digestive and Kidney Diseases; Neurons; Nuclear; Nuclear Extract; Nuclear Proteins; Outcome Study; Pattern; Phase; Phase Transition; Physics; Polymers; Precipitation; Procedures; Process; Proteins; RNA; RNA Polymerase II; RNA polymerase II largest subunit; RNA-Binding Proteins; Reaction; Reagent; Relative (related person); Sampling; Science; Serine; Stress; Structure; Surface; TATA-Box Binding Protein; Testing; Tissue Extracts; Transcriptional Activation; Transcriptional Regulation; Trinucleotide Repeats; Tyrosine; Work; X ray diffraction analysis; X-Ray Diffraction; Zinc Fingers; bHLH Domain; base; genetic regulatory protein; in vivo; medical schools; member; polyglutamine; polymerization; polypeptide; red fluorescent protein; research study; sarcoma; solid state nuclear magnetic resonance; transcription factor

DESCRIPTION (provided by applicant): For upwards of 30 years it has been known that regulatory proteins involved in the control of gene expression and mRNA biogenesis contain low complexity (LC) polypeptide sequences that are intrinsically disordered. In the case of gene specific transcription factors, these LC sequences, often typified by long, homopolymeric segments of polyglutamine, have been implicated in the illusive process of transcriptional activation. It is well-understood how the DNA binding domains of transcription factors, including zinc fingers, leucine zippers, bHLH domains, and homeobox domains, facilitate direct and specific interaction between transcription factors and their target genes. By contrast, the field remains largely ignorant of the mechanisms by which the LC sequences typifying "activation domains" facilitate gene expression. In the case of proteins that regulate RNA biogenesis, similarly perplexing LC sequences are often linked to well-folded domains that mediate direct interaction with RNA substrates (including RRM, KH and pumlio domains). By serendipity, members of the McKnight laboratory discovered that a biotinylated isoxazole (b-isox) chemical is capable of reversibly co-precipitating hundreds of RNA binding proteins from cytosolic extracts. This unexpected discovery formed the starting point for experiments showing that LC sequences can reversibly polymerize into cross-beta filaments that phase transition to a hydrogel-like state. In turn, microscopic versions of these hydrogel droplets were evolved into a simplified, quantitative assay for gel retention of homotypic and heterotypic test proteins. By discovering that repeats of the sequence [G/S]Y[G/S] serve as a nucleating substrate for fiber polymerization, it was possible to test the correlative effects of mutational impediments to fiber polymerization in vitro with the ability of RNA binding proteins to move in and out of RNA granules in living cells. As a result of this work, it has been hypothesized that reversible polymerization of LC domains may represent the organizational basis for the formation of RNA granules including P-granules, stress granules, P- bodies and neuronal granules (Kato et al., 2012; Han et al., 2012). As an extension of this work nuclear extracts have been exposed to the b-isox chemical. These experiments led to the reversible precipitation of many nuclear proteins containing LC sequences, including TATA binding protein, TAF15, the largest subunit of RNA polymerase II, and components of the mediator complex. A major objective of the experiments proposed in this grant application will be the test of whether similar processes are employed to control the organization of nuclear puncta, including Cajal bodies, nuclear speckles and "transcription factories".

描述（由申请人提供）：已知超过30年，众所周知，与控制基因表达和mRNA生物发生有关的调节蛋白含有固有无序无序的低复杂性（LC）多肽序列。对于基因特异性转录因子，这些LC序列通常以多谷氨酰胺的长长，均聚糖段为代表，与转录激活的虚幻过程有关。转录因子的DNA结合结构域（包括锌指，亮氨酸拉链，BHLH结构域和同源蛋黄域）如何促进转录因子及其靶基因之间的直接和特定相互作用。相比之下，该场在很大程度上仍然不了解lc序列典型的“激活结构域”促进基因表达的机制。在调节RNA生物发生的蛋白质中，类似的困惑LC序列通常与围绕与RNA底物直接相互作用（包括RRM，KH和PUMLIO结构域）直接相互作用的折叠结构域相关。通过偶然性，McKnight实验室的成员发现，生物素化的氧唑（B-ISOX）化学能够从胞质提取物中可逆地共沉淀数百种RN​​A结合蛋白。这种意外的发现构成了实验的起点，表明LC序列可以可逆地聚合到相结合丝中，并将相变到类似水凝胶状状态。反过来，将这些水凝胶液滴的显微镜版本进化为简化的定量测定，用于凝胶保留同型和异型测试蛋白。通过发现序列的重复[g/s] y [g/s]是纤维聚合的成核底物，可以测试突变障碍在体外与纤维聚合与RNA结合蛋白的能力在活细胞中移动和脱离RNA的能力。这项工作的结果是，已经假设LC结构域的可逆聚合可能代表形成RNA颗粒的组织基础，包括P颗粒，应激颗粒，P-体和神经元颗粒（Kato等，2012; Han等，2012; Han等，2012）。作为这项工作的扩展，核提取物已暴露于B-ESOX化学物质。这些实验导致许多含有LC序列的核蛋白的可逆沉淀，包括TATA结合蛋白，TAF15，TAF15，RNA聚合酶II的最大亚基和介体复合物的成分。在本赠款申请中提出的实验的一个主要目标是测试是否采用了类似的过程来控制核点的组织，包括Cajal机构，核斑点和“转录工厂”。