Experimental identification of microRNA targets in mosquitoes during Plasmodium i

说明（由申请人提供）：2009年全球报告了2.43亿例疟疾病例和86.3万例归因于疟疾的死亡。为了从一个人传播到另一个人，疟疾寄生虫（疟原虫）必须在蚊子体内完成一个复杂的发育程序，并在蚊子先天免疫系统的攻击下存活下来。我们的长期目标是发现蚊子参与抗疟原虫防御的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。microrna是大多数真核生物中的小内源性调节分子。在动物中，成熟的microrna被整合到microrna诱导的沉默复合体（miRISC）中，并与特定靶mRNA的3'非翻译区相关联，通过抑制翻译或mRNA降解来抑制基因表达。在冈比亚按蚊中，一些microrna的水平受到疟原虫感染的显著影响。rnai介导的Dicer-1或Argonaute-1 （microRNA通路的关键成分）的沉默已被证明可以提高寄生虫的存活率。因此，我们假设microrna在调节蚊子对疟疾寄生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，这些mrna只有在蚊子接触到血液中的疟疾寄生虫后才会被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的精确靶点。本项目的具体目的是：(1)确定在恶性疟原虫入侵过程中富集miRISC的冈比亚按蚊microrna。(2)鉴定受microrna选择性调控的蚊子信使rna，以应对疟原虫的侵袭。我们提出的研究解决了在蚊子-疟原虫相互作用中对microRNA功能理解的严重空白，并可能为阻断疟疾传播提供新的分子靶点。

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.