Reversible Polymerization of Low Complexity Polypeptide Sequences as a Framework

描述（由申请人提供）：30多年来，人们已经知道，参与控制基因表达和mRNA生物发生的调节蛋白含有低复杂性（LC）多肽序列，这些序列本质上是无序的。在基因特异性转录因子的情况下，这些LC序列，通常以长，聚谷氨酰胺的同聚片段为典型，涉及转录激活的虚幻过程。转录因子的DNA结合域，包括锌指结构域、亮氨酸拉链结构域、bHLH结构域和同源盒结构域，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，已经得到了很好的理解。相比之下，该领域在很大程度上仍然对LC序列“激活域”促进基因表达的机制一无所知。在调节RNA生物发生的蛋白质中，同样令人困惑的LC序列通常与介导与RNA底物直接相互作用的折叠良好的结构域（包括RRM， KH和pumlio结构域）相关联。偶然的机会，麦克奈特实验室的成员发现一种生物素化的异恶唑（b-isox）化学物质能够从细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，表明LC序列可以可逆地聚合成交叉β丝，并相转变为水凝胶状状态。反过来，这些水凝胶液滴的微观版本演变为一种简化的定量分析，用于检测同型和异型测试蛋白的凝胶保留。通过发现序列[G/S]Y[G/S]的重复序列作为纤维聚合的成核底物，可以在体外测试突变障碍对纤维聚合的相关影响，以及RNA结合蛋白在活细胞中进出RNA颗粒的能力。由于这项工作，人们假设LC结构域的可逆聚合可能代表了RNA颗粒形成的组织基础，包括P-颗粒、应力颗粒、P-体和神经元颗粒（Kato et al., 2012; Han et al., 2012）。作为这项工作的延伸，核提取物已暴露于b-异染色体化学。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、TAF15 （RNA聚合酶II的最大亚基）和介质复合物的组分。这项拨款申请中提出的实验的一个主要目标是测试是否采用类似的过程来控制核点的组织，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".