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

逃避宿主免疫反应的共同需求导致了进化出非常相似的生存策略，即使在进化上遥远的生物体之间也是如此。其中一种策略是抗原变异——生物体周期性地改变向宿主免疫系统显示的蛋白质身份的能力。在许多利用抗原变异机制来逃避宿主免疫反应的感染性微生物中，一些最大的相互排他表达抗原家族存在于原生动物寄生虫中，如恶性疟原虫、贾第鞭毛虫和人类昏睡病的病原体布鲁氏锥虫。布鲁氏体基因组编码约2500个变异表面糖蛋白（VSGs）同工型。大约1000万个相同的VSGs拷贝形成一个致密的表面涂层，保护不变的寄生虫蛋白不被宿主免疫反应识别。不同VSG异构体的互斥表达以及从一种VSG异构体到另一种VSG异构体的周期性表达转换允许寄生虫改变其表面涂层组成，从而逃避宿主的免疫反应。虽然对布鲁氏T.的抗原变异已经有了很多了解，但导致一种VSG的转录抑制和另一种VSG亚型的激活的分子机制仍然是难以捉摸的。最近在恶性疟原虫和G. lamblia中的发现表明，抗原变异的严格调控受到特异性非编码rna （ncRNA）存在与否的影响。因此，我们决定研究ncRNA在布鲁氏体抗原变异中的作用。随着布鲁氏体rna测序和核糖体分析技术的建立，使我们能够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的同一区域包含一个对VSG连续表达至关重要的“稳定元件”，该区域的短缺失导致VSG切换频率的强烈增加。基于我们的发现和ncRNA在其他原生动物寄生虫中确保互斥表达的重要性，我们假设新发现的长ncRNA是布鲁氏T.的抗原变异所必需的。这项工作的目的是验证这一假设，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，可以操纵vsg基因上游区域等调控元件-这是本文工作成功的关键特征。

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.