Identification and function of the VSG transcript-bound proteome

VSG转录结合蛋白质组的鉴定和功能

基本信息

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
项目类别：
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

项目摘要

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. ! !

项目概要布氏锥虫引起人类和牲畜的非洲锥虫病。血流型 (BF) 阶段，布氏锥虫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，有可能转换为不同的 VSG 基因，这是一种现象这使得寄生虫能够逃避宿主免疫系统的作用并被称为抗原变异。导致慢性感染 BF 锥虫细胞表面覆盖有约 1100 万个 VSG 蛋白，它可以由约 1,400 个 VSG mRNA 拷贝（约占总 mRNA 的 7%）翻译而来。对于 BF T. brucei 的生存至关重要，寄生虫必须维持 VSG mRNA 的功能性高水平，并且 VSG 蛋白在 T. brucei 中以多顺反子方式转录，其中包括 VSG 一种方式。实现高水平的 VSG mRNA 是通过转录表达单个 VSG 等位基因。转录活性“BF 表达位点 (BES)”，一种多顺反子转录单元 (PTU)，含有 RNA 存在 pol I 启动子、几个表达位点相关基因 (ESAG) 和一个 VSG。但只有一种具有转录活性，而其余的 BES 则受到抑制，尽管 ESAG 水平很高。和 VSG 转录本是由活性 BES 产生的，丰度差异显着，与 VSG 转录本比 ESAGs 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。最有可能的是维持高水平 VSG mRNA 的 RNA 结合蛋白 (RBP)。尽管在 BF 阶段表达，但在 T. brucei 分化过程中生命周期的其他阶段不表达。尽管很重要，但关于 VSG mRNA 转录后调控的机制几乎一无所知，除了嵌入 VSG mRNA（16 聚体）3'UTR 中的短保守序列元件赋予两个阶段在这里，我们发现 VSG mRNA 的 3´UTR 具有序列特异性结合。 RBP 的位点以及该 VSG mRNA 核糖核蛋白颗粒“VSG”的组装和组成 mRNPs’，促进翻译和/或保护 VSG mRNA 免遭降解，维持大量 VSG mRNA 以及蛋白质在本提案中，我们的目标是鉴定 BF T. brucei 中 VSG mRNP 的成分。我们将首先从基因和生物化学角度鉴定目标 1 中 VSG mRNP 复合物的成分，以及验证目标 2 中的潜在候选者。VSG 在它可以保护的 BF 锥虫的存活中发挥多种作用；尽管 VSG 是一种强抗原，但也能引发强烈的适应性宿主免疫反应。由于抗原变异，开发非洲锥虫病疫苗疗法一直很困难。 VSG 对于 BF T. brucei 绝对重要，了解 VSG 表达控制机制（特别是 RBP 如何影响 VSG mRNP 的功能）不仅使我们能够发现基本原理布氏锥虫基因表达的生物学，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。！！