RNA BINDING PROTEINS IN PLASMODIUM FALCIPARUM

恶性疟原虫中的 RNA 结合蛋白

• 批准号: 2887700
• 负责人: Laurie K. Read
• 金额: $ 7.7万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-09-30 至 2001-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887700
• 关键词: Plasmodium falciparum; RNA binding protein; affinity chromatography; complementary DNA; gene expression; genetic library; immunoprecipitation; nucleic acid sequence; polymerase chain reaction; posttranscriptional RNA processing; protein localization; protein structure function

Plasmodium falciparum, the causative agent of the most virulent form of human malaria, is responsible for between one and two million deaths per year. Since the recent reemergence P. falciparum is in large part a result of increased drug resistance, understanding of drug resistance mechanisms and identification of novel drug targets are urgent goals. Of fundamental importance to both of these goals is an increased knowledge of the mechanisms regulating gene expression in P. falciparum. Several observations indicate that posttranslational regulation of gene expression plays a role at multiple levels in this organism, including mRNA splicing, polyadenylation, cleavage of polycistronic precursor mRNAs, and mRNA stability. However, little is known about the details of these processes in P. falciparum. RNA binding proteins have been shown to be involved in all of these processes in other organisms. This proposal is designed to identify RNA binding proteins in P. falciparum and to begin to assess their functions in this organism. We will use a multi-faceted approach to isolate RNA binding proteins and their genes. This approach will include PCR amplification using primers corresponding to conserved RNA binding domains, screening of cDNA expression libraries with both single- and double-stranded RNA probes, and direct biochemical purification of abundant RNA binding proteins by affinity chromatography. Full length cDNA sequences will then be obtained by 5' and 3' RACE or by screening cDNA libraries. Once clones encoding RNA binding proteins are in hand, the proteins will be expressed and antibodies produced. Developmental regulation and subcellular localization will be assessed to provide information regarding the proteins' functions. General RNA binding preferences will be determined in competition experiments. Specific cellular RNA targets will be identified using an immunoprecipitation approach and/or by iterative selection from a random RNA library (SELEX). These experiments will identify the first RNA binding proteins from P. falciparum, and provide evidence regarding the function of the proteins in the regulation of P. falciparum gene expression. The information obtained regarding gene expression may prove relevant to our understanding of drug resistance mechanisms. Moreover, since genetic mechanisms in lower eukaryotes are frequently divergent, novel proteins identified during this study could provide new targets for chemotherapeutic intervention in malaria.

恶性疟原虫，最致命的恶性疟原虫的病原体， 人类疟疾每年造成100万至200万人死亡， 年 自从最近重新出现恶性疟原虫， 耐药性增加的结果，了解耐药性 机制和鉴定新的药物靶点是迫切的目标。 对这两个目标至关重要的是， 了解恶性疟原虫基因表达调控机制。 一些观察结果表明，基因的翻译后调节 表达在该生物体中的多个水平发挥作用，包括 mRNA剪接、多聚腺苷酸化、多顺反子前体切割 mRNA和mRNA稳定性。 然而，有关细节知之甚少 恶性疟原虫的这些过程。 RNA结合蛋白已被 参与了其他生物体的所有这些过程。 这 一项旨在鉴定恶性疟原虫RNA结合蛋白的提案 并开始评估它们在这个生物体中的功能。 我们将使用 一种分离RNA结合蛋白及其 基因. 该方法将包括使用引物的PCR扩增 对应于保守的RNA结合结构域，筛选cDNA 具有单链和双链RNA探针的表达文库， 以及通过以下方法直接生物化学纯化丰富的RNA结合蛋白： 亲和层析 然后将全长cDNA序列 通过5 ′和3 ′ RACE或通过筛选cDNA文库获得。 一旦克隆 编码RNA结合蛋白的蛋白质在手，蛋白质将被 表达并产生抗体。 发展监管和 将评估亚细胞定位以提供信息 关于蛋白质的功能。 一般RNA结合偏好将 在竞争实验中确定。 特异性细胞RNA靶标 将使用免疫沉淀方法和/或通过 从随机RNA文库迭代选择（SELEX）。这些实验 将鉴定出恶性疟原虫的第一个RNA结合蛋白， 提供了关于蛋白质在细胞中功能的证据。 恶性疟原虫基因表达的调控。 获得的信息 关于基因表达的研究可能与我们对 耐药机制 此外，由于遗传机制在低 真核生物经常是不同的，新的蛋白质鉴定期间， 这项研究可能为化疗干预提供新的靶点 在疟疾中。