Zhang Yang, Zhao Bo, Roy Sourav, Saha Tusar T, Kokoza Vladimir A, Li Ming, Raikhel Alexander S
Department of Entomology, University of California, Riverside, CA 92521; Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China;
Department of Entomology, University of California, Riverside, CA 92521; Institute for Integrative Genomic Biology, University of California, Riverside, CA 92521.
Proc Natl Acad Sci U S A. 2016 Aug 16;113(33):E4828-36. doi: 10.1073/pnas.1609792113. Epub 2016 Aug 3.
Obligatory blood-triggered reproductive strategy is an evolutionary adaptation of mosquitoes for rapid egg development. It contributes to the vectorial capacity of these insects. Therefore, understanding the molecular mechanisms underlying reproductive processes is of particular importance. Here, we report that microRNA-309 (miR-309) plays a critical role in mosquito reproduction. A spatiotemporal expression profile of miR-309 displayed its blood feeding-dependent onset and ovary-specific manifestation in female Aedes aegypti mosquitoes. Antagomir silencing of miR-309 impaired ovarian development and resulted in nonsynchronized follicle growth. Furthermore, the genetic disruption of miR-309 by CRISPR/Cas9 system led to the developmental failure of primary follicle formation. Examination of genomic responses to miR-309 depletion revealed that several pathways associated with ovarian development are down-regulated. Comparative analysis of genes obtained from the high-throughput RNA sequencing of ovarian tissue from the miR-309 antagomir-silenced mosquitoes with those from the in silico computation target prediction identified that the gene-encoding SIX homeobox 4 protein (SIX4) is a putative target of miR-309. Reporter assay and RNA immunoprecipitation confirmed that SIX4 is a direct target of miR-309. RNA interference of SIX4 was able to rescue phenotypic manifestations caused by miR-309 depletion. Thus, miR-309 plays a critical role in mosquito reproduction by targeting SIX4 in the ovary and serves as a regulatory switch permitting a stage-specific degradation of the ovarian SIX4 mRNA. In turn, this microRNA (miRNA)-targeted degradation is required for appropriate initiation of a blood feeding-triggered phase of ovarian development, highlighting involvement of this miRNA in mosquito reproduction.
必需的血液触发生殖策略是蚊子为实现快速卵子发育而进行的一种进化适应。它有助于这些昆虫的传播能力。因此，了解生殖过程背后的分子机制尤为重要。在此，我们报告微小RNA-309（miR-309）在蚊子繁殖中起关键作用。miR-309的时空表达谱显示其在雌性埃及伊蚊中依赖于血液摄入而开始表达，并在卵巢中特异性表现。miR-309的拮抗剂沉默会损害卵巢发育，并导致卵泡生长不同步。此外，通过CRISPR/Cas9系统对miR-309进行基因破坏会导致初级卵泡形成的发育失败。对miR-309缺失的基因组反应的检查表明，与卵巢发育相关的几个途径被下调。对来自miR-309拮抗剂沉默蚊子的卵巢组织的高通量RNA测序获得的基因与计算机计算目标预测获得的基因进行比较分析，确定编码SIX同源盒4蛋白（SIX4）的基因是miR-309的一个假定靶点。报告基因测定和RNA免疫沉淀证实SIX4是miR-309的直接靶点。SIX4的RNA干扰能够挽救由miR-309缺失引起的表型表现。因此，miR-309通过靶向卵巢中的SIX4在蚊子繁殖中起关键作用，并作为一个调节开关允许卵巢SIX4 mRNA进行阶段特异性降解。反过来，这种微小RNA（miRNA）靶向降解对于适当启动血液摄入触发的卵巢发育阶段是必需的，突出了这种miRNA在蚊子繁殖中的参与。
