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Processing of Potato Spindle Tuber Viroids (PSTVd) RNAs in Yeast, a Nonconventional Host

机译：非常规宿主酵母中马铃薯纺锤块茎类病毒（PSTVd）RNA的加工

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摘要

The discovery of viroids in 1971 opened the door to a whole new field of RNA biochemistry. Viroids subsequently became the first of many facets of RNA biochemistry: the first single stranded covalently closed RNA discovered in nature, the first subviral pathogen discovered, and the first pathogen of a eukaryotic system to have its genome sequenced. Viroids are the smallest known agents of infectious disease and they represent the borders of life. They replicate autonomously within their host and since they do not code for their own proteins, they act as scavengers of the host transcriptional machinery. By doing so, viroids find ways of trafficking, localizing, and replicating within their host based on the sequence and structure of the RNA alone. Once in their hosts, viroids are incredibly resilient and can cause economic damage on several commercial crops. Apart from controlling viroids for economic reasons, the more enticing feature of viroid study is the use of viroids as model systems to study essential underlying questions about the evolution of RNA pathogens, and to use viroids as models to study non-coding RNAs. The field of non-coding RNA research has surged within the past decade and viroids are becoming important vehicles to bring insight into this field of study. The study of viroids has been extensive through the years, but several questions remain: What structural conformations do viroids employ to recruit host enzymes, and what are the enzymes that cleave and ligate viroids into mature progeny. To answer some of these questions, we have looked at processing of the potato spindle tuber viroid (PSTVd) RNA in the budding yeast Saccharomyces cerevisiae. We found that one specific construct will process into a mature viroid circle in yeast and we also found that processing in this system is distinct from other plant and non-plant based host systems. This processing is a delicate interplay of ligation and degradation by host machinery. Yeast is a great system to study viroid processing as yeast allows for use of the entire toolbox of temperature-sensitive and knockout protein mutants. By employing yeast, focus can be driven towards the mechanisms of host protein recruitment, viroid processing requirements, and degradation mechanisms from the host. We have ascertained insight into PSTVd processing using yeast. We have found methods to transform and process PSTVd, investigated enzymes that effect processing, and started to establish an in vitro yeast system. Through these studies, we have also developed a method to enrich viroid RNAs from total RNA extractives. This has been vital to assays specific around viroid transcription and cleavage. Overall, this research is further testament that viroids are minimalist scavengers of a very diverse array of cellular transcriptional machinery. They can process in higher eukaryotes (plants) and simple eukaryotes (yeast). They are shown to affect each host in distinct manners using fundamental RNA biology that all organisms share.
机译：1971年类病毒的发现为RNA生物化学的新领域打开了大门。病毒随后成为RNA生物化学众多方面中的第一个：自然界中发现的第一个单链共价闭合RNA，第一个发现亚病毒的病原体以及第一个对其基因组进行测序的真核系统的病原体。类病毒是已知的最小的传染病媒介，它们代表生命的边界。它们在宿主内自主复制，并且由于它们不为自己的蛋白质编码，因此它们充当宿主转录机制的清除剂。通过这样做，类病毒会根据单独的RNA序列和结构找到在其宿主内进行运输，定位和复制的方式。一旦进入宿主，类病毒就会具有难以置信的弹性，并可能对几种商业作物造成经济损失。除了出于经济原因控制类病毒之外，类病毒研究的更诱人的功能是使用类病毒作为模型系统来研究有关RNA病原体进化的基本潜在问题，并使用类病毒作为模型来研究非编码RNA。在过去的十年中，非编码RNA的研究领域激增，类病毒正成为将洞察力带入这一研究领域的重要工具。多年来，对类病毒的研究一直很广泛，但是仍然存在几个问题：类病毒利用哪些结构构象来募集宿主酶，以及将类病毒裂解并连接成成熟后代的酶是什么。为了回答其中的一些问题，我们研究了芽孢酵母酿酒酵母中马铃薯纺锤块茎类病毒（PSTVd）RNA的加工过程。我们发现一种特定的构建体将在酵母中加工成成熟的类病毒环，并且我们还发现，该系统中的加工不同于其他基于植物和非植物的宿主系统。该处理是宿主机器对连接和降解的微妙相互作用。酵母是研究类病毒加工的绝佳系统，因为酵母允许使用温度敏感和敲除蛋白突变体的整个工具箱。通过使用酵母，可以将注意力转向宿主蛋白质募集的机制，类病毒加工的要求以及宿主的降解机制。我们已经确定了使用酵母处理PSTVd的见解。我们已经找到了转化和加工PSTVd的方法，研究了影响加工的酶，并开始建立体外酵母系统。通过这些研究，我们还开发了一种从总RNA提取物中富集类病毒RNA的方法。这对于围绕类病毒转录和裂解的特异性检测至关重要。总的来说，这项研究进一步证明了类病毒是多种多样的细胞转录机制的极简清除剂。它们可以加工成高等真核生物（植物）和简单的真核生物（酵母）。通过使用所有生物共有的基本RNA生物学，可以证明它们以不同的方式影响每个宿主。

著录项

  • 作者

    Friday, Dillon R.;

  • 作者单位

    University of the Sciences in Philadelphia.;

  • 授予单位 University of the Sciences in Philadelphia.;
  • 学科 Virology.;Molecular biology.;Biochemistry.
  • 学位 Ph.D.
  • 年度 2017
  • 页码 152 p.
  • 总页数 152
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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