Ribosome Profiling in Plasmodium falciparum

恶性疟原虫核糖体分析

• 批准号: 8661701
• 负责人: Peter John Myler
• 金额: $ 23万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-15 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8661701
• 关键词: Attention; Binding; Bioinformatics; Biological Assay; Blood; Code; Complex; Couples; Coupling; Culicidae; Data; Development; Developmental Process; Disease; Drug resistance; Erythrocytes; Exons; Future; Gene Expression; Generations; Genes; Genetic Translation; Genome; Goals; Heat-Shock Response; Human; Individual; Infection; Ingestion; Initiator Codon; Introns; Knowledge; Left; Leishmania; Libraries; Life; Life Cycle Stages; Liver; Location; Malaria; Measures; Mediating; Messenger RNA; Military Personnel; Morbidity - disease rate; Nutrient; Open Reading Frames; Organism; PAWR protein; Pancreatic ribonuclease; Parasites; Peptide Initiation Factors; Pharmaceutical Preparations; Phase; Phosphorylation; Phosphotransferases; Plasmodium; Plasmodium falciparum; Play; Pregnancy; Proteins; Proteome; Proteomics; RNA; RNA library; RNA-Binding Proteins; Ribosomes; Role; Saccharomyces cerevisiae; Seminal; Sexual Development; Signal Transduction; Specific qualifier value; Sporozoites; Staging; Stress; Structure; System; Technology; Testing; Time; Toxoplasma; Transcript; Translating; Translational Regulation; Translational Repression; Translations; Trypanosoma; Trypanosoma brucei brucei; Vaccines; Variant; Virus Diseases; Work; Yeasts; asexual; deep sequencing; environmental change; extracellular; fighting; genome annotation; improved; killings; mRNA Expression; new technology; next generation sequencing; nuclease; programs; public health relevance; research study; response; tool; transcriptome sequencing; transmission process

DESCRIPTION (provided by applicant): The malaria parasite Plasmodium falciparum kills over 650,000 people a year and causes widespread morbidity. Human infection commences with a silent phase in the liver, after which the parasites emerge into the blood to infect red blood cells. There, the parasites develop in an ordered, yet graded, progression from rings to trophozoites to schizonts before escaping as merozites to start another erythrocytic cycle and disease. During the erythrocytic cycle, a structured cycle of mRNA abundance is evident, but the correlation between protein and mRNA levels is, at best, modest. This project explores the hypothesis that translational regulation tunes the proteome in the face of a regimented program for mRNA expression. Recently work has shown that translational controls are likely to be important in the erythrocytic cycle, as well as in the transition from mosquito sporozoites to live stages and in the development of the sexual stages essential to transmission. A corollary of these studies is that gene products that are highly regulated at the level of translation are likel important modulators of developmental change and the parasite's response to environmental stress. The project goals are to globally and quantitatively assess the rate at which each mRNA is actively translated in specific erythrocytic stages by coupling the ability to isolate the speciic "footprints" of mRNAs that are occupied by ribosomes (an indicator of translation) with the depth and breadth of next generation sequencing. Aim 1 will establish the ribosome footprinting technology in P. falciparum using trophozoite stage parasites. It will optimize conditions for nuclease treatment to generate the mRNA footprints protected by ribosomes and for the generation of unbiased libraries from the RNA footprints for Illumina deep sequencing. It will also include maturation of the bioinformatics pipeline to analyze resulting sequence data, adapting existing pipelines that are used for RNA-Seq in P. falciparum and for ribosome footprinting of trypanosomes. Aim 2 will expand into other stages of the asexual erythrocytic cycle, identifying genes that are differentially translated across these pathogenic stages. It will also examine gametocytes, which are poised for an abrupt environmental change upon ingestion by the mosquito. The proposed work will yield both an overview of the extent of translational control in P. falciparum blood stages, as well as a quantification of the translationl control of individual gene products. From such data, additional hypotheses on mechanisms of control and functions of regulated proteins can be generated and tested. The project will also improve genome annotation by determining whether proteins are translated from mRNAs corresponding to individual open reading frames, particularly those annotated as hypothetical proteins and those encoded by non-canonical protein-coding open reading frames, to provide a comprehensive view of the translational landscape of P. falciparum erythrocytic stages.

描述（由申请人提供）：恶性疟原虫疟原虫每年杀死650,000多人，并引起广泛的发病率。人类感染从肝脏中的沉默阶段开始，此后，寄生虫出现进入血液以感染红细胞。在那里，寄生虫以有序但分级的进展从环向滋养体到岩岩的进展发展，然后逃脱为梅罗齐特人开始了另一个红细胞周期和疾病。在红细胞周期中，mRNA丰度的结构化循环是显而易见的，但是蛋白质和mRNA水平之间的相关性充其量是适度的。该项目探讨了这样一个假设，即翻译调节会在面对mRNA表达的条例程序中调节蛋白质组。最近的工作表明，翻译控制可能在红细胞周期以及从蚊子孢子岩到现场阶段和发展对传播必不可少的性阶段的发展中很重要。这些研究的推论是，在翻译层面高度调节的基因产物可能是发育变化的重要调节剂，以及寄生虫对环境压力的反应。项目目标是通过耦合将核糖体占据的特定“足迹”与下一代测序深度和广度分离出来的特定的mRNA的特定“足迹”，以在全球范围内和定量评估每个mRNA在特定的红细胞阶段积极翻译的速率。 AIM 1将使用滋养体阶段寄生虫在恶性疟原虫中建立核糖体足迹技术。它将优化核酸酶处理的条件，以产生受核糖体保护的mRNA足迹，并从RNA足迹中生成无偏见的文库，以进行Illumina深层测序。它还将包括生物信息学管道的成熟，以分析所得的序列数据，调整用于恶性疟原虫中RNA-Seq的现有管道和用于锥虫的核糖体足迹。 AIM 2将扩展到无性红细胞周期的其他阶段，鉴定在这些致病阶段差异翻译的基因。会 还要检查蚊子在摄入时突然改变环境变化的配子细胞。拟议的工作将概述恶性疟原虫血液阶段的翻译控制程度，以及对单个基因产物的翻译控制的定量。从这些数据中，可以生成和测试受调节蛋白质的控制机制和功能的其他假设。该项目还将通过确定是否从与单个开放式阅读框相对应的mRNA转换来改善基因组注释，尤其是那些被注释为假设蛋白的蛋白质，以及那些由非典型蛋白质蛋白质编码开放式阅读框架编码的蛋白质，以便全面地观察到卵巢层甲状腺疟原虫的翻译景观。