Identification and function of the VSG transcript-bound proteome

VSG转录结合蛋白质组的鉴定和功能

• 批准号: 10250512
• 负责人: Esteban Daniel Erben
• 金额: $ 19.88万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10250512
• 关键词: Actins; Affect; African Trypanosomiasis; Alleles; Antigenic Variation; Antigens; Area; B-Lymphocytes; Binding; Binding Proteins; Binding Sites; Biochemical; Biological Assay; Biological Models; Biology; Blood Circulation; Catalogs; Categories; Cell Cycle; Cell Survival; Cells; Complex; Consequentialism; Conserved Sequence; DNA Polymerase I; Data Set; Development; Disease; Elements; Flow Cytometry; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genetic Translation; Half-Life; High-Throughput Nucleotide Sequencing; Homeostasis; Human; Immune response; Immune system; Immunoglobulins; Lead; Libraries; Life Cycle Stages; Livestock; Maintenance; Mass Spectrum Analysis; Membrane Glycoproteins; Messenger RNA; Mutation; Open Reading Frames; Outcome; Parasites; Population; Post-Transcriptional Regulation; Protein Overexpression; Proteins; Proteome; Publishing; RNA; RNA Binding; RNA Interference; RNA interference screen; RNA-Binding Proteins; Regulation; Reporter; Research; Ribonucleoproteins; Role; Site; Specificity; Surface; Surface Antigens; System; Testing; Therapeutic Intervention; Transcript; Translating; Translations; Trypanosoma; Trypanosoma brucei brucei; Vaccine Therapy; Variant; candidate validation; chronic infection; effective therapy; experimental study; genetic approach; genetic variant; human disease; human pathogen; insight; mRNA Stability; mRNA Transcript Degradation; messenger ribonucleoprotein; neglect; novel; novel therapeutic intervention; overexpression; particle; promoter; protein complex; tool

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. ! !

项目总结 布氏锥虫会在人和牲畜中引起非洲锥虫病。在哺乳动物中感染性 布鲁氏毛滴虫(T.brucei)血流形成期(BF)表达单种变异型表面糖蛋白(VSG) 来自2500多个VSG谱系的基因有可能切换到不同的VSG基因，这是一种现象 被称为抗原性变异。这使得寄生虫能够逃脱宿主免疫系统的作用， 会导致慢性感染。BF锥虫细胞的表面覆盖着大约1100万个VSG蛋白， 它可以从大约1,400个拷贝的VSG mRNA(约占总mRNA的7%)翻译而来。因为VSG是 对于BF T.brucei的生存至关重要，寄生虫必须保持VSG mRNA和VSG的功能性高水平 VSG蛋白才能存活。几乎所有的基因在布氏毛滴虫中都是多顺反子转录的，包括VSG。单程 要获得高水平的VSG mRNA是通过转录实现的。一个VSG等位基因从 转录活性的BF表达位点(BES)，一个含有RNA的多聚柠檬酸转录单位(PTU) POL I启动子、几个表达位点相关基因(ESAGs)和一个VSG。大约有15名女演员出席 但只有一个是转录活跃的，其余的BEs被抑制。尽管ESAG水平很高 而VSG转录本是由活性的BES产生的，丰度与VSG转录本有显著差异 比ESAG高100~1000倍。这表明一定存在反式作用的VSG mRNA特异性调节因子， 最有可能的是维持VSG高水平的RNA结合蛋白(RBP)。此外，VSG是 在布氏锥虫分化的BF期表达，而在生活史的其他阶段不表达。尽管 重要的是，对VSG mRNA转录后调控的机制几乎一无所知，除了 VSG mRNA3‘非编码区(16-mer)中嵌入的一个短的、保守的序列元件使这两个阶段 特异性和稳定性。在这里，我们假设VSG基因的3‘非编码区具有序列特异性结合 限制性商业惯例的位点，以及这种VSG mRNA核糖核蛋白颗粒的组装和组成 MRNPs，促进翻译和/或保护VSG mRNA免受降解，维持VSG的大量 因此，蛋白质也是如此。在这项建议中，我们的目标是确定BF·布氏锥虫中VSG mRNPs的成分。 我们将首先从遗传学和生物化学的角度鉴定目标1中VSG mRNP复合体的成分 验证AIM 2中的潜在候选者。VSG在BF锥虫的生存中扮演多种角色；它可以保护 这种寄生虫还能引发强烈的适应性宿主免疫反应。尽管VSG是一种很强的抗原， 由于抗原的多样性，开发非洲锥虫病的疫苗疗法一直很困难。vt.给出 VSG对于BF T.brucei来说是绝对必要的，了解VSG的表达控制机制 (具体地说，限制性商业惯例如何影响VSG mRNP的功能)不仅使我们能够发现 布鲁氏毛滴虫的生物学基因表达，同时也发现了一种可能导致治疗干预的新生物学 这种人类疾病。 好了！ 好了！