Ribosome Profiling in Plasmodium falciparum

描述（由申请人提供）：疟疾寄生虫恶性疟原虫每年导致超过65万人死亡，并引起广泛的发病率。人类感染开始于肝脏的一个沉默阶段，之后寄生虫进入血液感染红细胞。在那里，寄生虫以有序而有序的方式发展，从环体到滋养体再到分裂体，然后以分裂体的形式逃脱，开始另一个红细胞周期和疾病。在红细胞周期中，mRNA丰度的结构周期是明显的，但蛋白质和mRNA水平之间的相关性充其量是适度的。该项目探讨了翻译调控在面对mRNA表达调控程序时调节蛋白质组的假设。最近的研究表明，翻译控制可能在红细胞周期中很重要，也可能在从蚊子孢子体到活体阶段的过渡中很重要，也可能在传播所必需的性阶段的发展中很重要。这些研究的一个推论是，在翻译水平上受到高度调控的基因产物可能是发育变化和寄生虫对环境应激反应的重要调节剂。该项目的目标是通过将分离被核糖体（翻译指标）占据的mRNA的特定“足迹”的能力与下一代测序的深度和广度相结合，全面和定量地评估每个mRNA在特定红细胞阶段活跃翻译的速率。目的1利用滋养体阶段寄生虫建立恶性疟原虫核糖体足迹技术。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足迹中生成用于Illumina深度测序的无偏文库。它还将包括成熟的生物信息学管道，以分析所得到的序列数据，调整现有的用于恶性疟原虫RNA-Seq和锥虫核糖体足迹的管道。目的2将扩展到无性红细胞周期的其他阶段，确定在这些致病阶段差异翻译的基因。它将

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.