Unraveling the role of trypanosomal ncRNA in the regulation of antigenic variation

揭示锥虫 ncRNA 在抗原变异调节中的作用

• 批准号: 277883612
• 负责人: Professor Dr. Tim Nicolai Siegel
• 金额: --
• 依托单位: Experimentelle Parasitologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2018-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/277883612?language=en
• 关键词: Unraveling; role; trypanosomal; ncRNA; regulation

The common need to evade the host immune response has led to the evolution of remarkably similar survival strategies even among evolutionarily distant organisms. One of these strategies is antigenic variation - the ability of an organism to periodically change the identity of the proteins displayed to the host immune system. Among the many infectious microorganisms that utilize mechanisms of antigenic variation to evade the host immune response some of the largest families of mutually exclusively expressed antigens are found in protozoan parasites such as Plasmodium falciparum, Giardia lamblia and Trypanosoma brucei, the causative agent of human sleeping sickness. The T. brucei genome codes for ~2500 isoforms of variant surface glycoproteins (VSGs). Roughly 10 million identical copies of VSGs form a dense surface coat that shields invariant parasite proteins from recognition by the host immune response. The mutually exclusive expression of different VSG isoforms and the periodic switch in expression from one VSG isoform to another permit the parasite to change its surface coat composition and, as a consequence, to evade the host immune response. While much has been learned about antigenic variation in T. brucei, the molecular mechanism leading to the transcriptional repression of one VSG and activation of another VSG isoform has remained elusive. Recent findings in P. falciparum and G. lamblia suggest that the tight regulation of antigenic variation is affected by the presence or absence of specific non-coding RNAs (ncRNA). Therefore, we have decided to investigate the role of ncRNA in antigenic variation in T. brucei. Following the establishment of RNA-sequencing and ribosome-profiling technology in T. brucei, which allowed us to perform genome-wide transcriptome and translatome analyses, we searched the genome for transcripts not translated into protein, i.e. putative ncRNA. Our data revealed that the region upstream of the actively transcribed vsg is transcribed into long ncRNA. Intriguingly, more than 15 years ago it was found that the same region that we find to be transcribed into long ncRNA contains a 'stabilizing element' crucial for continuous VSG expression and that short deletions in this region lead to a strong increase in VSG switching frequency. Based on our findings and the importance of ncRNA in ensuring mutually exclusive expression in other protozoan parasites, we hypothesize that the newly identified long ncRNA is required for antigenic variation in T. brucei. The goal of this work is to test this hypothesis and to elucidate the role of the region upstream of the vsg in stabilizing VSG expression. Taking advantage of the recently established CRISPR/Cas9 technology, we are for the first time able to perform marker-free genome-editing in T. brucei putting us in the unique position to manipulate regulatory elements like the region upstream of the vsg gene - a feature crucial for the success of the work proposed here.

逃避宿主免疫反应的共同需要导致了即使在进化上相距遥远的生物体中也进化出非常相似的生存策略。其中一种策略是抗原变异——生物体定期改变向宿主免疫系统展示的蛋白质特性的能力。在许多利用抗原变异机制逃避宿主免疫反应的传染性微生物中，一些最大的相互排斥表达抗原家族存在于原生动物寄生虫中，例如恶性疟原虫、贾第鞭毛虫和布氏锥虫（人类昏睡病的病原体）。 T. brucei 基因组编码约 2500 种变异表面糖蛋白 (VSG) 亚型。大约 1000 万个相同的 VSG 拷贝形成致密的表面涂层，保护不变的寄生虫蛋白不被宿主免疫反应识别。不同 VSG 同工型的相互排斥表达以及从一种 VSG 同工型表达到另一种的周期性转换允许寄生虫改变其表面涂层组成，从而逃避宿主免疫反应。虽然人们对布氏锥虫的抗原变异了解很多，但导致一种 VSG 转录抑制和另一种 VSG 亚型激活的分子机制仍然难以捉摸。最近在恶性疟原虫和兰氏疟原虫中的发现表明，抗原变异的严格调节受到特定非编码 RNA (ncRNA) 存在或不存在的影响。因此，我们决定研究 ncRNA 在布氏锥虫抗原变异中的作用。在 T. brucei 中建立 RNA 测序和核糖体分析技术后，我们可以进行全基因组转录组和翻译组分析，我们在基因组中搜索未翻译成蛋白质的转录本，即假定的 ncRNA。我们的数据显示，活跃转录的 vsg 上游区域被转录成长 ncRNA。有趣的是，超过 15 年前，我们发现转录成长 ncRNA 的同一区域包含对连续 VSG 表达至关重要的“稳定元件”，并且该区域的短缺失会导致 VSG 转换频率的大幅增加。根据我们的发现以及 ncRNA 在确保其他原生动物寄生虫中相互排斥表达方面的重要性，我们假设新鉴定的长 ncRNA 是布氏锥虫抗原变异所必需的。这项工作的目的是检验这一假设并阐明 vsg 上游区域在稳定 VSG 表达中的作用。利用最近建立的 CRISPR/Cas9 技术，我们首次能够在 T. brucei 中进行无标记基因组编辑，使我们处于独特的位置来操纵调节元件，如 vsg 基因上游区域 - 这是本文提出的工作成功的关键特征。