Experimental identification of microRNA targets in mosquitoes during Plasmodium i

描述（由申请人提供）：2009年全球报告了2.43亿例疟疾病例和863，000例归因死亡。疟疾寄生虫（Plasmodium spp.）必须在蚊子体内完成一个精心设计的发育程序，并在蚊子先天免疫系统的攻击下生存下来。我们的长期目标是发现参与抗疟原虫防御的蚊子microRNA，阐明这些microRNA的分子功能，并利用这些知识来帮助未来控制疟疾传播的努力。 microRNA是大多数真核生物中的内源性小分子调节分子。在动物中，成熟的microRNA被整合到microRNA诱导的沉默复合物（miRISC）中，并与特定靶mRNA的3'非翻译区结合，以通过抑制翻译或通过mRNA降解来抑制基因表达。在冈比亚按蚊中，疟原虫感染显著影响中肠中某些microRNA的水平。RNAi介导的Dicer-1或Argonaute-1（microRNA途径的关键组分）的沉默已显示出增强寄生虫存活。因此，我们假设microRNA在调节蚊子对疟原虫的防御反应中起重要作用。 为了验证这一假设，我们需要找到蚊子的mRNA，只有在蚊子暴露于血液中的疟疾寄生虫后，它们才能被microRNA特异性调节。仅通过计算方法预测microRNA靶标通常受到高假阳性率的阻碍。我们将使用HITS-CLIP（通过交联免疫沉淀分离的RNA的高通量测序）方法来捕获和鉴定miRISC中Argonaute-1结合的microRNA和靶mRNA。这将揭示中肠中表达的所有信使RNA的精确靶位点。本项目的具体目标是：（1）确定恶性疟原虫入侵过程中miRISC中富集的冈比亚按蚊microRNA。(2)识别蚊子信使RNA，选择性地调节microRNA响应疟原虫的挑战。 我们提出的研究解决了对蚊子-疟原虫相互作用中microRNA功能理解的严重差距，并可能为阻断疟疾传播提供新的分子靶点。 公共卫生相关性：雌性按蚊是人类疟疾的传播媒介。蚊子体内的一些小RNA分子参与调节蚊子对疟原虫的防御反应。我们的研究将确定这些小RNA的靶点，而这些见解反过来可能会导致疟疾控制的新策略。

DESCRIPTION (provided by applicant): 243 million malaria cases and 863,000 attributed deaths were reported globally in 2009. To be transmitted from one person to another, the malaria parasite (Plasmodium spp.) has to complete an elaborate developmental program in the mosquito and survive the attacks from mosquito innate immune system. Our long term goal is to discover mosquito microRNAs that are involved in anti-Plasmodium defense, elucidate the molecular functions of these microRNAs, and to use this knowledge to aid future efforts to control malaria transmission. MicroRNAs are small endogenous regulatory molecules in most eukaryotes. In animals, mature microRNAs are integrated into a microRNA-induced silencing complex (miRISC) and associated with the 3' untranslated regions of specific target mRNAs to suppress gene expression either through inhibition of translation or through mRNA degradation. In Anopheles gambiae mosquitoes, levels of some microRNAs are markedly affected in the midgut by Plasmodium infection. RNAi-mediated silencing of either Dicer-1 or Argonaute-1, key components of the microRNA pathway, has been shown to enhance parasite survival. We therefore hypothesize that microRNAs play an important role in modulating mosquito defense response to malaria parasites. To test this hypothesis, we need to find mosquito mRNAs that are specifically regulated by microRNAs only after mosquitoes are exposed to malaria parasites in a blood meal. microRNA target prediction by computational approaches alone is generally hampered by high false-positive rates. We will use the HITS-CLIP (high-throughput sequencing of RNAs isolated by crosslinking immunoprecipitation) method instead to capture and identify both the microRNAs and target mRNAs bound by Argonaute-1 in miRISC. This will reveal precise target sites across all messenger RNAs expressed in the midgut. The specific aims of this project are to: (1) Determine Anopheles gambiae microRNAs that are enriched in miRISC during Plasmodium falciparum invasion. (2) Identify mosquito messenger RNAs that are selectively regulated by microRNAs in response to Plasmodium challenge. Our proposed studies address a serious gap in the understanding of microRNA functions in the mosquito-Plasmodium interactions, and may provide novel molecular targets for blocking malaria transmission. PUBLIC HEALTH RELEVANCE: The female Anopheles mosquito is the vector for human malaria. Some small RNA molecules in the mosquito are implicated in regulating the mosquito defense reactions against malaria parasite. Our research will identify the targets of those small RNAs and the insights in turn may lead to new strategies for malaria control.