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. ! !

项目摘要布鲁氏锥虫引起人类和牲畜的非洲锥虫病。在哺乳动物的传染性血流形式（BF）阶段，T。Brucei表达了一种变异表面糖蛋白（VSG）的单一种类从2500多个VSG曲目中的基因，有可能切换到不同VSG基因的基因，这是一种现象称为抗原变异。这使寄生虫可以摆脱宿主免疫系统的作用，并导致慢性感染。 BF锥虫细胞的表面涂有约1100万个VSG蛋白，可以从约1,400份VSG mRNA（约占总mRNA的7％）翻译。因为VSG是 BF T. Brucei的生存能力至关重要，寄生虫必须保持功能高水平的VSG mRNA和 VSG蛋白生存。几乎所有基因在T. brucei（包括VSG）中均以多余的方式转录。单程为了达到高水平的VSG mRNA是通过转录。一个VSG等位基因从转录活跃的“ BF表达位点（BES）”，一个多柱转录单元（PTU），包含RNA Pol I启动子，几种表达位点相关基因（ESAGS）和A VSG。大约有15个贝斯但是只有一个是转录活跃的，其余的贝丝被反映了。虽然高水平的ESAG VSG转录本是由活性BES产生的，抽象与VSG转录物显着区分比ESAG高100〜1000倍。这表明必须有反式vsg mRNA特定调节剂，最有可能维持高水平VSG mRNA的RNA结合蛋白（RBP）。另外，VSG是在BF阶段表达，但在T. brucei分化期间的其他阶段不表达。尽管有重要的是，关于VSG mRNA的转录后调节机制几乎一无所知，除了那个简短的构成序列元素嵌入了VSG mRNA（16-MER）的3´UTR中，供认了这两个阶段特异性和稳定性。在这里，我们假设VSG mRNA的3´UTR具有序列特异性结合 RBP的位点以及该VSG mRNA核糖核蛋白颗粒的组装和组成，'VSG mRNPS，促进翻译和/或保护VSG mRNA免于降解，维持大量VSG mRNA，因此也是蛋白质。在此提案中，我们旨在确定BF T. Brucei中VSG mRNP的组成部分。我们将在AIM 1中首先从遗传和生物化学上确定VSG MRNP复合物的成分在AIM 2中验证潜在的候选物。VSG在BF锥虫的存活中起多种作用。它可以保护寄生虫但也可以引发强大的适应性宿主免疫反应。即使VSG是一种强大的抗原，由于抗原差异，开发针对非洲锥虫病的疫苗疗法一直很困难。给出该VSG对于BF T. Brucei绝对必要，了解VSG表达控制机制（特别是RBP如何对VSG mRNP的功能的影响）不仅可以使我们发现基本布鲁氏菌基因表达的生物学，但也发现一种新的生物学，可以导致治疗干预这种人类疾病。呢呢