Dengue virus infection alters posttranscriptional modification of microRNAs in the mosquito vector Aedes aegypti

Recent discoveries regarding the importance of isomiRs have increased our understanding of the regulatory complexities of the miRNAome. Observed changes in the miRNA profiles in mosquitoes infected with flaviviruses have implicated small RNAs in the interactions between viruses and their vectors. Here we analysed the isomiR profiles of both uninfected and infected Aedes aegypti mosquitoes with the major human pathogen dengue virus (DENV). We found that several specific isomiRs were significantly altered in their abundance patterns in response to DENV infection potentially affecting their target repertoire. Notable among these were isomiR variants which displayed arm-switching. We also demonstrate that modifications to the 3p end of miRNAs are vastly more prevalent than those at the 5p ends. We also observed that in only 45% of Ae. aegypti miRNAs the most abundant read matches the exact sequence reported in miRBase. Further, we found positive correlations between the number of mature miRNA reads, pre-miRNA length, GC content and secondary structure minimum free energy with the number of isomiRs. The findings presented here provide some evidence that isomiR production is not a random phenomenon and may be important in DENV replication in its vector

Wolbachia-induced aae-miR-12 miRNA negatively regulates the expression of MCT1 and MCM6 genes in Wolbachia-infected mosquito cell line

Background: Best recognized for its role in manipulating host reproduction, the parasitic gram-negative Wolbachia pipientis is known to colonize a wide range of invertebrates. The endosymbiotic bacterium has recently been shown to cause a life-shortening effect as well as inhibiting replication of arboviruses in Aades aegypti; although the molecular mechanisms behind these effects are largely unknown. MicroRNAs (miRNAs) have been determined to have a wide range of roles in regulating gene expression in eukaryotes. A recent study showed that several A. aegypti mosquito miRNAs are differentially expressed when infected with Wolbachia

Inhibition of aae-miR-12 affects <i>Wolbachia</i> density.

<p>qPCR analysis of DNA isolated from aag2-<i>w</i>MelPop-CLA cells mock-transfected, transfected with the control inhibitor or aae-miR-12 inhibitor. The error bars indicate standard deviations of averages from two biological and three technical replicates. There are no statistical significant differences within the group with the same letter at <i>p</i>&gt;0.05.</p

The <i>A. aegypti MCM6</i>, <i>Exoculease</i> and <i>MCT1</i> were predicted to be the best targets of aae-miR-12 with significant sequence complementarities.

<p>The <i>A. aegypti MCM6</i>, <i>Exoculease</i> and <i>MCT1</i> were predicted to be the best targets of aae-miR-12 with significant sequence complementarities.</p

Validation of aae-miR-12 interaction with <i>MCT1</i> and <i>MCM6</i> target genes using miRNA mimics.

<p>(<b>A</b>) qRT-PCR analysis of RNA extracted from Aag2 cells mock-transfected and those transfected with the aae-miR-12 mimic or the control mimic (C-mimic). Specific primers to the <i>MCM6</i> were used. (<b>B</b>) Similar experiment as in (A) repeated with mutant mimic 1 (mut-1) and 2 (mut-2) of aae-miR-12. (<b>C</b>) qRT-PCR analysis of RNA extracted from Aag2 cells mock-transfected and those transfected with the aae-miR-12 mimic or the control mimic (C-mimic). Specific primers to the <i>MCT1</i> were used. (<b>D</b>) Similar experiment as in (C) repeated with mutant mimic 1 (mut-1) and 2 (mut-2) of aae-miR-12. The error bars in A–D indicate standard deviations of averages from two biological and three technical replicates. There are no statistical significant differences within the group with the same letter at <i>p</i>&gt;0.05.</p

qRT-PCR analysis of predicated target genes of aae-miR-12 in aag2-<i>w</i>MelPop-CLA and uninfected Aag2 cells.

<p>The error bars indicate standard deviations of averages from two biological and three technical replicates. ***, <i>p</i>&lt;0.001; ns, <i>p</i>&gt;0.05.</p

Predicted targets of aae-miR-12.

<p>Predicted targets of aae-miR-12.</p

In vivo effect of <i>Wolbachia</i> on <i>MCM6</i> and <i>MCT1</i> transcript levels.

<p>qRT-PCR conducted on total RNA extracted from whole female mosquitoes. Specific primers were used for (<b>A</b>) <i>MCM6</i> and (<b>B</b>) <i>MCT1</i> to determine transcript levels in mosquitoes infected with <i>Wolbachia</i> (Wol+) as well as those not infected (Wol−). The error bars indicate the standard deviation in the averages of 3 biological and 3 technical replicates. The cumulative tissue from ten whole mosquitoes (4 days old) was used for each biological replicate. ***, <i>p</i>&lt;0.001; ns, not significant <i>p</i>&gt;0.05.</p

qRT-PCR analysis of RNA extracted from Sf9 cells co-transfected with either (A) pIZ-GFP-<i>MCM6</i> (pIZ/MCM6) or (B) pIZ-GFP-<i>MCT1</i> (pIZ/MCT1) constructs and control mimic (C-mimic) or aae-miR-12 mimic.

<p>The error bars indicate standard deviations of averages from two biological and three technical replicates. There are no statistical significant differences within the group with the same letter at <i>p</i>&gt;0.05.</p