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

逃避宿主免疫反应的共同需要导致了非常相似的生存策略的进化，即使在进化上遥远的生物体中也是如此。这些策略之一是抗原变异-生物体周期性改变向宿主免疫系统展示的蛋白质身份的能力。在利用抗原变异机制逃避宿主免疫应答的许多感染性微生物中，在原生动物寄生虫如恶性疟原虫、蓝氏贾第鞭毛虫和布氏锥虫（人类昏睡病的病原体）中发现了一些最大的互斥表达抗原家族。霸王布鲁氏菌基因组编码约2500种变体表面糖蛋白（VSG）的同种型。大约有1000万个相同的VSG拷贝形成了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同种型的互斥表达和从一种VSG同种型到另一种VSG同种型的表达的周期性切换允许寄生虫改变其表面被衣组成，并且因此逃避宿主免疫应答。虽然对T.然而，导致一种VSG的转录抑制和另一种VSG同种型的激活的分子机制仍然难以捉摸。恶性疟原虫（P.falciparum）和G. Lamblia的研究表明，抗原变异的严格调节受到特异性非编码RNA（ncRNA）存在与否的影响。因此，我们决定研究ncRNA在T.布鲁塞。随着T.布氏杆菌，这使我们能够进行全基因组转录组和翻译组分析，我们搜索基因组中未翻译成蛋白质的转录物，即推定的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连续VSG表达至关重要的“稳定元件”，并且该区域的短缺失导致VSG转换频率的强烈增加。基于我们的发现和ncRNA在确保其他原生动物寄生虫中相互排斥表达的重要性，我们假设新鉴定的长ncRNA是T.布鲁塞。这项工作的目标是测试这一假设，并阐明该地区的vsg上游稳定VSG表达的作用。利用最近建立的CRISPR/Cas9技术，我们首次能够在T. brucei使我们处于独特的位置来操纵调控元件，如vsg基因上游区域，这是本文提出的工作成功的关键。

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.