Experimental identification of microRNA targets in mosquitoes during Plasmodium i

描述(申请人提供)：2009年，全球共报告了2.43亿疟疾病例和863,000例归因性死亡。从一个人传给另一个人的疟疾寄生虫(疟疾属)必须在蚊子体内完成一个复杂的发育程序，并在蚊子固有免疫系统的攻击下存活下来。我们的长期目标是发现参与抗疟原虫防御的蚊子microRNAs，阐明这些microRNAs的分子功能，并利用这一知识帮助未来控制疟疾传播的努力。在大多数真核生物中，microRNAs是一种小的内源性调节分子。在动物中，成熟的microRNAs被整合到microRNA诱导的沉默复合体(MiRISC)中，并与特定靶标mRNAs的3‘非翻译区结合，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，一些microRNAs的水平受到疟原虫感染的显著影响。RNAi介导的沉默是microRNA途径的关键组成部分，Dice-1或ArgAerte-1，已被证明可以提高寄生虫的存活率。因此，我们假设microRNAs在调节蚊子对疟疾寄生虫的防御反应中发挥重要作用。为了验证这一假说，我们需要找到只有在蚊子接触到血餐中的疟疾寄生虫后，才能被microRNA特异性调控的蚊子mRNAs。仅靠计算方法预测microRNA靶标通常会受到高假阳性率的阻碍。我们将使用HITS-CLIP(交联式免疫沉淀分离的RNA高通量测序)方法来捕获和鉴定miRISC中与ArgAerte-1结合的microRNAs和靶mRNAs。这将揭示中肠表达的所有信使RNA的准确靶点。本项目的具体目标是：(1)确定恶性疟原虫入侵过程中富含miRISC的冈比亚按蚊microRNAs。(2)鉴定蚊子信使RNA是由microRNAs选择性调控的，以响应疟原虫攻击。我们提出的研究解决了在蚊子-疟原虫相互作用中对microRNA功能的理解的一个严重差距，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媒介。蚊子中的一些小RNA分子参与调节蚊子对疟疾寄生虫的防御反应。我们的研究将确定这些小RNA的靶标，这些见解反过来可能会导致疟疾控制的新战略。

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.