Biochemical & Genetic Analysis of Low Complexity Domains in RNA-binding protein biology

摘要/摘要许多参与基因表达的蛋白质都含有一个低复杂性结构域， 本身缺乏二级结构，但可以形成分子间聚合物，称为淀粉样蛋白，一种 稳定的结构，可以组织成细丝和水凝胶。一些携带这种物质的蛋白质 固有的非结构结构域与RNA聚合酶II或在 抄写。事实上，这种结构特征在细胞RNA结合中被异常地过度表达 蛋白质。目前的一个假说表明，这些低复杂性结构域是蛋白质类的。 开关，与其相邻的折叠部分，如RNA识别基序，组装成细胞 处理和处理核糖核酸的隔间。很好的例子包括酵母NaB3和Nrd1，它 是终止sRNA和snoRNAs等短转录本所需的因子。同样， 潜在的淀粉样蛋白形成结构域，将mRNA多聚腺苷化偶联到末端终止 蛋白质编码基因见于Pcf11、Hrp1和Rat1。低复杂度自组装域 与人类疾病有因果关系，特别是神经退行性病理 异常的细胞内含物是一个共同的特征。一个主要的模型是，正常的倾向 因为RNA结合蛋白的聚集可能会出错，导致有毒的低聚物和聚合体 与细胞损坏相关的程序集。然而，我们对这一现象的理解很差。 这些结构域的正常可逆功能以及为什么它们的聚集会变得有毒。 该项目的长期目标是了解淀粉样蛋白形成区域提供了什么 核糖核酸结合蛋白的功能；尤其是那些参与终止核糖核酸转录的蛋白质 聚合酶II。工作假说是这些蛋白质的条件聚合是一个关键 这是他们运作方式的一部分。一种模型是，这些蛋白质将新生RNA包裹在特定的 提供给诸如解旋酶和解旋酶等终止和加工的酶的方式 核酸酶；也许是以核小体包装DNA的方式。这项工作的重点是RNA结合， 酵母中参与转录终止的转录终止因子 这一点我很理解。所有这些都具有低复杂性的结构域，可能会发生齐聚。这个 原型是酵母hnRNP样蛋白NaB3，它带有富含多聚谷氨酰胺的自组装 结构域对细胞活性至关重要，对转录终止也很重要。此项目测试 这些结构域在转录终止机制中的作用涉及：1)它们的淀粉样蛋白 形成潜力，2)细胞存活的需要，3)终止的要求。 这些目标的成功将导致关于这些RNA-蛋白质相互作用如何的新的机制模型 带来高效和准确的基因表达。

Summary/Abstract Many proteins involved in gene expression contain a low complexity domain that itself lacks secondary structure but which can form intermolecular polymers known as amyloid, a stable structure that can organize into filaments and hydrogels. Some of the proteins that bear such intrinsically unstructured domains interact with RNA polymerase II or the RNA produced during transcription. Indeed, this structural feature is unusually over-represented in cellular RNA- binding proteins. A current hypothesis suggests that these low complexity domains are proteinaceous switches that, with their adjacent folded portions such as RNA recognition motifs, assemble into cellular compartments that handle and process RNA. Excellent examples include yeast Nab3 and Nrd1 which are factors needed for termination of short transcripts such as snRNAs and snoRNAs. Similarly, potential amyloid forming domains that couple mRNA polyadenylation to termination at the end of protein coding genes are seen in Pcf11, Hrp1, and Rat1. Low complexity self-assembling domains are causally implicated in human diseases, particularly neurodegenerative pathologies in which aberrant cellular inclusions are a common characteristic. A leading model is that the normal propensity for aggregation of RNA-binding proteins can go awry, leading to toxic oligomeric and polymeric assemblies that are associated with cellular damage. Yet we have a poor understanding of the normal reversible function of these domains and why their aggregation can become toxic. The long-term goal of the project is to understand what amyloid forming domains provide for the function of RNA binding proteins; particularly those involved in terminating transcription of RNA polymerase II. The working hypothesis is that conditional polymerization of these proteins is a key part of how they operate. One model is that these proteins enshroud nascent RNA in a specific manner for presentation to the enzymes of termination and processing such as helicases and nucleases; perhaps in the way nucleosomes package DNA. The focus of this work is on RNA- binding, transcription termination factors in yeast where their involvement in the termination of transcription is fairly well understood. All possess low complexity domains that can potentially oligomerize. The prototype is the yeast hnRNP-like protein Nab3 that bears a polyglutamine-rich, self-assembly domain essential for cell viability and important for transcription termination. This project tests the role of these domains in the transcription termination machinery with respect to: 1) their amyloid forming potential, 2) the need for them for cell viability, and 3) their requirement for termination. Success in these aims will lead to new mechanistic models of how these RNA-protein interactions lead to productive and accurate gene expression.