Reversible Polymerization of Low Complexity Polypeptide Sequences as a Framework

描述（由申请人提供）：30多年来，已知参与控制基因表达和mRNA生物发生的调节蛋白含有本质上无序的低复杂性（LC）多肽序列。在基因特异性转录因子的情况下，这些LC序列，通常以多聚谷氨酰胺的长的均聚片段为代表，已经涉及转录激活的虚幻过程。转录因子的DNA结合结构域（包括锌指、亮氨酸拉链、bHLH结构域和同源框结构域）如何促进转录因子与其靶基因之间的直接和特异性相互作用是众所周知的。相比之下，该领域在很大程度上仍然不知道代表“激活结构域”的LC序列促进基因表达的机制。在调节RNA生物发生的蛋白质的情况下，类似地令人困惑的LC序列通常连接到介导与RNA底物直接相互作用的良好折叠的结构域（包括RRM、KH和pumlio结构域）。McKnight实验室的成员偶然发现，一种生物素化的异恶唑（b-isox）化学物质能够从胞质提取物中可逆地共沉淀数百种RNA结合蛋白。这一意想不到的发现形成了实验的起点，该实验表明LC序列可以可逆地折叠成相变为水凝胶样状态的交叉β丝。反过来，这些水凝胶液滴的显微镜版本演变成用于同型和异型测试蛋白质的凝胶保留的简化的定量测定。通过发现序列[G/S]Y[G/S]的重复作为纤维聚合的成核底物，可以测试体外纤维聚合的突变障碍与RNA结合蛋白移入和移出活细胞中RNA颗粒的能力的相关影响。作为这项工作的结果，已经假设LC结构域的可逆聚合可以代表RNA颗粒（包括P-颗粒、应激颗粒、P-小体和神经元颗粒）形成的组织基础（Kato et al. 2012; Han等人，2012年）。作为这项工作的延伸，核提取物已暴露于b-isox化学品。这些实验导致了许多含有LC序列的核蛋白的可逆沉淀，包括TATA结合蛋白、TAF 15、RNA聚合酶II的最大亚基和介体复合物的组分。在这项资助申请中提出的实验的一个主要目标将是测试是否采用类似的过程来控制核斑点的组织，包括卡哈尔体，核斑点和“转录工厂”。

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".