Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
Transcriptome-wide microRNA and target dynamics in the fat body during the gonadotrophic cycle of Aedes aegypti
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DOI：
10.1073/pnas.1701474114
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发表时间：
2017-02
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影响因子：
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通讯作者：
Xiufeng Zhang;E. Aksoy;T. Girke;A. Raikhel;Fedor V. Karginov
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作者：
Xiufeng Zhang;E. Aksoy;T. Girke;A. Raikhel;Fedor V. Karginov
意义控制蚊子疾病媒介传播的一个潜在途径在于血餐后的生殖事件。一个关键组成部分是在脂肪体组织中大量生产蛋黄蛋白，由可用营养素触发的调节网络控制。microRNA在蚊子卵成熟中起着关键作用，并且破译它们的动力学和靶点对于充分实现这些调控过程是必要的。我们对埃及伊蚊脂肪体中microRNA表达进行了组织特异性和时间分辨的表征，并将这些结果与其mRNA靶点的转录组范围确定相结合，然后进行验证。这种广泛的分析为系统地了解支持雌性蚊子生殖事件的基因调控奠定了基础。埃及伊蚊是许多病毒性疾病的主要媒介，因为它需要血粉来促进卵的发育。脂肪体是哺乳动物肝脏和脂肪组织的对应物，是代谢中心，在生殖中起着关键作用。因此，了解控制其功能的调控网络至关重要，而microRNAs（miRNAs）在这一过程中的作用在很大程度上是未知的。我们的目的是探索miRNA在雌性Ae脂肪体中的表达和潜在靶点。埃及，以及他们的变化postblood餐（PBM）。小RNA文库分析揭示了在五个采样时间点顺序表达的五种独特的miRNA模式，可能响应并影响同一时期上游激素信号和基因表达的波动。为了将miRNA身份与下游靶点联系起来，在封闭后72 h和PBM 24 h通过Argonaute 1交联和免疫沉淀，随后通过高通量测序，实验确定了转录组范围的mRNA 3′ UTR相互作用位点。通过体外荧光素酶试验，用野生型和突变的3′ UTR对6个miRNA家族的几个靶位点进行了验证。对于已建立的转基因系，通过时空敲减miR-8和荧光素酶测定观察到一致的结果。我们进一步研究了可能调节各种生理过程的miRNA，这些过程基于Orthopathological Groups功能类别的簇。因此，本研究全面阐明了雌蚊脂肪体中miRNA的表达和靶点动态，为进一步研究miRNA在促性腺周期中的调控功能奠定了基础。
Significance A potential avenue to control the spread of mosquito disease vectors lies in reproductive events that follow a blood meal. A key component is the massive production of yolk proteins in the fat body tissue, governed by regulatory networks triggered by the available nutrients. MicroRNAs play a critical role in mosquito egg maturation, and deciphering their dynamics and targets is necessary to fully realize these regulatory processes. We carried out a tissue-specific and time-resolved characterization of microRNA expression in the Aedes aegypti fat body and integrated these results with transcriptome-wide determination of their mRNA targets, followed by validation. This extensive analysis lays the groundwork for a systemic understanding of the gene regulation that underpins reproductive events in the female mosquito. The mosquito Aedes aegypti is a major vector of numerous viral diseases, because it requires a blood meal to facilitate egg development. The fat body, a counterpart of mammalian liver and adipose tissues, is the metabolic center, playing a key role in reproduction. Therefore, understanding of regulatory networks controlling its functions is critical, and the role of microRNAs (miRNAs) in the process is largely unknown. We aimed to explore miRNA expression and potential targets in the female fat body of Ae. aegypti, as well as their changes postblood meal (PBM). Small RNA library analysis revealed five unique miRNA patterns sequentially expressed at five sampled time points, likely responding to, and affecting, waves of upstream hormonal signals and gene expression in the same period. To link miRNA identities with downstream targets, transcriptome-wide mRNA 3′ UTR interaction sites were experimentally determined at 72 h posteclosion and 24 h PBM through Argonaute 1 cross-linking and immunoprecipitation followed by high-throughput sequencing. Several target sites were validated by means of in vitro luciferase assays with wild-type and mutated 3′ UTRs for six miRNA families. With established transgenic lines, consistent results were observed with spatiotemporal knockdown of miR-8 and luciferase assays. We further investigated miRNAs potentially regulating various physiological processes based on Clusters of Orthologous Groups functional categories. Hence, the present work comprehensively elucidated miRNA expression and target dynamics in the female mosquito fat body, providing a solid foundation for future functional studies of miRNA regulation during the gonadotrophic cycle.