Fig 1.
CLEAR-CLIP uncovers miRNA-mRNA interactions in An. gambiae.
(A) Key events during the first gonadotrophic cycle of An. gambiae. (B) CLEAR-CLIP generated both Ago1 CLIP reads and miRNA-target chimeric reads. Clustering of the CLIP reads disclosed Ago1 binding sites. (C) Distributions of all miRNA-target chimeras and Ago1 CLIP reads in various transcript regions. Other RNAs included introns and intergenic transcripts. PE, post eclosion. PBM, post blood meal. (D) Density plot of canonical miRNA seed matches in target mRNAs relative to the ligation site in all chimeras. In the shuffled interactions (gray), each chimeric target region was randomly re-assigned to a different miRNA. (E) Predicted minimum free energy between miRNA and target mRNA found in chimeras. The shuffled interactions served as control. The difference was calculated using the median of minimum free energy predicted by RNAhybrid. (F) Examples of the miR-309-SIX4 chimeric reads. The green dash line represents miR-309 and the red lines denote overlapping SIX4 sequences discovered in the chimeras. The miRNA-target duplex was predicted using RNAhybrid. (G) Mapping of Ago1 CLIP reads (orange) and the miR309-SIX4 chimeric reads (red) to the 3’ UTR of SIX4.
Fig 2.
Experimental validation of the chimera-defined targets of miR-309.
(A) Fold change in mRNA abundance in the ovary at 24 h PBM in An. gambiae injected with an antagomir for miR-309 verse a control antagomir. Antagomirs were injected into female adults at 2 h PE. The graph shows a cumulative distribution of the log2 fold change of mRNA levels for different sets of mRNAs: all targets identified in the chimeras involving miR-309 (red line), chimeras-defined miR-309 targets that were also supported by overlapping Ago1 CLIP peaks (blue line), and random transcripts which were not identified as the targets of miR-309 (black line). The difference between the distributions was analyzed by a Kolmogorov-Smirnov test. (B) Change in ribosome association of mRNA in An. gambiae after injection of antagomir-309. A scrambled antagomir sequence was used as a negative control. Ribosome occupancy was determined using ribosome footprinting. (C) Comparison of translational efficiency (TE) in An. gambiae injected with antagomir-309 or control antagomir. The TE of an mRNA was determined as ribosome occupancy normalized by its mRNA abundance. (D) Verification of the miR-309-target interactions using luciferase reporter assays. The reporter plasmids were constructed by inserting DNA fragments that contain the miR-309 target sites downstream of the Renilla luciferase coding region in a psiCHECK-2 vector. PCR primers used for cloning were listed in S5 Table. Results are shown as the ratio of the Renilla luciferase activity to the firefly luciferase activity (mean ± SD, n = 3). Statistical analyses were performed using a Student’s t-test (*, p<0.05; **, p<0.01; ***, p<0.001).
Fig 3.
Modulation of kr-h1 expression by let-7 is essential for normal egg development.
(A) The interactions of kr-h1 with let-7 and miR-2 defined by chimeric reads. (B) miRNA target sites in kr-h1. Distribution of Ago1 CLIP peaks (orange) was plotted along the kr-h1 transcript. Overlapped miRNA-target chimeras were labeled in red. (C) Luciferase reporter assays to test the interaction of miR-2, let-7 with kr-h1. DNA fragments bearing putative binding sites were cloned into psiCHECK-2. Three biological replicates were used for measurements. Statistical analyses were performed using a Student’s t-test (*, p<0.05; **, p<0.01). (D) Expression of Kr-h1 after antagomir injection. Specific (Ant-let-7, Ant-miR-2) and control (Ant-NC) antagomirs were injected into adult female mosquitoes at 12 h after adult emergence. Western blot was performed to measure the Kr-h1 protein at 120 h PE (before taking a blood meal) and at 24 h PBM after antagomir treatment. Non-inj, the uninjected control. (E) Oocyte development in the antagomir-treated mosquitoes at 24 h PBM. (F) Follicle sizes of the antagomir-treated mosquitoes at 24 h PBM. Thirty individuals in each group were measured using the Leica Application Suite (v4.5). Statistical analyses were performed using a Student’s t-test.
Fig 4.
Sequence motifs enriched in miRNA targets.
(A) Significantly enriched motifs for individual miRNAs were derived from chimeras-defined target sequences using the MEME tool. The MEME analysis was performed with the default 0-order Markov model to define 7-mer motifs. Shown here are motifs identified for representative miRNAs. N, number of motifs found/total number of targets analyzed; e-val, e-value of the motif reported by MEME. Seed sequences of miRNAs are in green. (B) De novo analysis of cognate miRNA-complementary-enriched 7-mer motifs in the chimeras that were detected at different time points. miRNAs with at least 30 unique chimeras-defined targets at a single time point were selected for the motif usage analysis using Homer. The motifs were plotted as a heatmap across the miRNA, with color intensity indicating the proportion of motifs in target sequences.
Fig 5.
Massive and dynamic miRNA-target interactions in mosquito reproduction.
(A) Patterns of miRNA-target interactions. The chimeras-supported and normalized Ago1CLIP peaks were analyzed using Gene Expression Dynamics Inspector (GEDI). The interaction data were clustered and transformed into a two-dimensional self-organizing map for individual time points. Each tile at the same position in all the maps corresponds to a group of miRNA-target interactions that share similar patterns throughout the 5 time points during adult reproduction. The color scale denotes the heights of Ago1 CLIP peaks. The small square represents the group that includes let-7-kr-h1 interaction. (B) Functional annotation of miRNA targets at each time point. (C) Regulation of mosquito metabolic genes by miRNAs in adult reproduction. The clustering analysis displays discrete patterns of interactions between mosquito miRNAs and genes involved in metabolism. The heights of peaks were z-normalized.
Fig 6.
Potential roles of miRNAs in the regulation of carbohydrate, lipid and amino acid metabolism in adult mosquitoes.
The chimeras-supported and normalized Ago1CLIP peaks were analyzed. The heatmaps show the patterns of miRNA interactions involving various metabolic genes. Dendrograms generated by hierarchical clustering of target genes from representative pathways are also provided. The full names of selected target genes are described in S6 Table.