Identification and function of the VSG transcript-bound proteome

项目摘要 布氏锥虫在人类和牲畜中引起非洲锥虫病。在哺乳动物传染性 BF期、T.布氏杆菌表达单一种类的变异表面糖蛋白（VSG） 来自超过2,500个VSG库的基因具有切换到不同VSG基因的潜力，这是一种现象， 称为抗原变异。这使得寄生虫能够逃避宿主免疫系统的作用， 导致慢性感染BF锥虫细胞的表面包被有约1100万个VSG蛋白， 其可以从约1，400个拷贝的VSG mRNA（约占总mRNA的7%）翻译。因为VSG是 对BF T的生存能力至关重要。布氏杆菌，寄生虫必须维持VSG mRNA的功能性高水平， VSG蛋白质来维持生命。在T.布鲁氏菌，包括VSG。一种方式 VSG mRNA的高表达是通过转录实现的。一个VSG等位基因表达于 转录活性的“BF表达位点（BES Ⅲ）”，一种多柠檬酸转录单位（PTU），含有RNA pol I启动子、几个表达位点相关基因（ESAG）和一个VSG。大约有15个BES存在 但只有一个是转录活性的，其余的BES被抑制。虽然ESAG的高水平 和VSG转录本是由活性BES产生的，丰度显著不同，VSG转录本 比ESAG高100~1000倍这表明必须存在反式作用的VSG mRNA特异性调节剂， 最可能的是维持VSG mRNA高水平的RNA结合蛋白（RBP）。此外，VSG 在T.布氏分化尽管 重要的是，几乎没有什么是已知的VSG mRNA的转录后调控机制，除了 嵌入VSG mRNA 3 ′ UTR的一个短的保守序列元件（16聚体）赋予了两个阶段 特异性和稳定性。在此我们假设VSG mRNA的3 ′ UTR具有序列特异性结合 RBP的位点，并且该VSG mRNA核糖核蛋白颗粒的组装和组成， mRNP“，促进翻译和/或保护VSG mRNA免于降解，维持大量的VSG mRNA， mRNA和蛋白质也是如此。在这个提议中，我们的目标是确定BF T中VSG mRNP的组分。布鲁塞。 我们将首先从遗传学和生物化学上鉴定Aim 1中VSG mRNP复合物的组分， 验证目标2中的潜在候选人。VSG在BF锥虫的生存中起着多重作用;它可以保护BF锥虫的生存， 寄生虫，但也可以触发强大的适应性宿主免疫反应。尽管VSG是一种强抗原， 由于抗原变异，开发非洲锥虫病的疫苗疗法是困难的。给定 VSG对于BF测试是绝对必要。了解VSG表达调控机制 （特别是RBP如何影响VSG mRNP的功能）不仅使我们能够发现基本的 生物学的T.布氏杆菌基因的表达，而且还发现了一种新的生物学， 这种人类疾病。 ! !

PROJECT SUMMARY Trypanosoma brucei causes African trypanosomiasis in humans and livestock. In the mammalian infectious Bloodstream-Form (BF) stage, T. brucei expresses a single species of Variant Surface Glycoprotein (VSG) genes from over 2,500 VSG repertoire with a potential of switching to a different VSG gene, a phenomenon known as antigenic variation. This allows the parasite to escape from the action of the host immune system and leads to a chronic infection. The surface of a BF trypanosome cell is coated with about 11 million VSG proteins, which can be translated from about 1,400 copies of VSG mRNA (about 7% of total mRNA). Because VSG is essential for the viability of BF T. brucei, the parasites must maintain the functional high level of VSG mRNA and VSG protein to survive. Almost all genes are transcribed polycistronically in T. brucei, including VSG. One way to achieve the high level of VSG mRNA is through transcription. A single VSG allele is expressed from the transcriptionally active ‘BF Expression Site (BES)’, a Polycitronic Transcription Unit (PTU), containing an RNA pol I promoter, several of Expression-Site Associated Genes (ESAGs) and a VSG. About 15 BESs are present but only one is transcriptionally active and the remaining BESs are repressed. Although high levels of ESAGs and VSG transcripts are produced from the active BES, the abundance differs significantly, with VSG transcripts 100~1000-fold higher than ESAGs. This suggests that there must be trans-acting VSG mRNA specific regulators, most probably RNA-binding proteins (RBPs) that maintain the high level of VSG mRNA. Additionally, VSG is expressed during BF stage but not in other stages of life-cycle during T. brucei differentiation. Despite the importance, almost nothing is known about mechanisms for post-transcriptional regulation of VSG mRNA, except that a short, conserved sequence element embedded into the 3´UTR of VSG mRNA (16-mer) confers both stage specificity and stability. Here we hypothesize that the 3´UTR of VSG mRNA have sequence-specific binding sites for RBPs and that the assembly and composition of this VSG mRNA RiboNucleoprotein Particles, ‘VSG mRNPs’, promote translation and/or protect VSG mRNA from degradation, maintaining the vast amount of VSG mRNA and thus, protein also. In this proposal we aim to identify the components of VSG mRNPs in BF T. brucei. We will first genetically and biochemically identify the components of the VSG mRNP complex in Aim 1 and validate potential candidates in Aim 2. VSG serves multiple roles in the survival of BF trypanosome; it can protect the parasite but can also trigger strong adaptive host immune response. Even though VSG is a strong antigen, developing vaccine therapy for African trypanosomiasis has been difficult due to the antigenic variation. Given that VSG is absolutely essential for BF T. brucei, understanding the VSG expression control mechanisms (specifically how RBPs impact on functions of VSG mRNPs) would not only enable us to discover fundamental biology of T. brucei gene expression but also to discover a new biology that could lead to therapeutic intervention of this human disease. ! !