Direct sequencing and expression analysis of a large number of miRNAs in Aedes aegypti and a multi-species survey of novel mosquito miRNAs

Abstract

Background: MicroRNAs (miRNAs) are a novel class of gene regulators whose biogenesis involves hairpin structures called precursor miRNAs, or pre-miRNAs. A pre-miRNA is processed to make a miRNA:miRNA* duplex, which is then separated to generate a mature miRNA and a miRNA*. The mature miRNAs play key regulatory roles during embryonic development as well as other cellular processes. They are also implicated in control of viral infection as well as innate immunity. Direct experimental evidence for mosquito miRNAs has been recently reported in anopheline mosquitoes based on small-scale cloning efforts.

Results: We obtained approximately 130, 000 small RNA sequences from the yellow fever mosquito, Aedes aegypti, by 454 sequencing of samples that were isolated from mixed-age embryos and midguts from sugar-fed and blood-fed females, respectively. We also performed bioinformatics analysis on the Ae. aegypti genome assembly to identify evidence for additional miRNAs. The combination of these approaches uncovered 98 different pre-miRNAs in Ae. aegypti which could produce 86 distinct miRNAs. Thirteen miRNAs, including eight novel miRNAs identified in this study, are currently only found in mosquitoes. We also identified five potential revisions to previously annotated miRNAs at the miRNA termini, two cases of highly abundant miRNA* sequences, 14 miRNA clusters, and 17 cases where more than one pre-miRNA hairpin produces the same or highly similar mature miRNAs. A number of miRNAs showed higher levels in midgut from blood-fed female than that from sugar-fed female, which was confirmed by northern blots on two of these miRNAs. Northern blots also revealed several miRNAs that showed stage-specific expression. Detailed expression analysis of eight of the 13 mosquito-specific miRNAs in four divergent mosquito genera identified cases of clearly conserved expression patterns and obvious differences. Four of the 13 miRNAs are specific to certain lineage(s) within mosquitoes.

Conclusion: This study provides the first systematic analysis of miRNAs in Ae. aegypti and offers a substantially expanded list of miRNAs for all mosquitoes. New insights were gained on the evolution of conserved and lineage-specific miRNAs in mosquitoes. The expression profiles of a few miRNAs suggest stage-specific functions and functions related to embryonic development or blood feeding. A better understanding of the functions of these miRNAs will offer new insights in mosquito biology and may lead to novel approaches to combat mosquito-borne infectious diseases.

Figure 1

Alignments and stem-loop structures of five novel mosquito pre-miRNAs. See Table 1 for naming and sequence locations of these miRNAs. Left panels are the hairpin structures. Right panels are the sequence alignments between Ae. aegypti miRNAs (aae-miRNAs) and Cx. quinquefasciatus miRNAs (cqu-miRNAs). Arrows point to the mature miRNA sequences from 5' to 3'. In the case of miR-M1, there are two physically linked copies in both Ae. aegypti and Cx. quinquefasciatus, as shown in the alignment and in Table 1. The two copies in Ae. aegypti produce the same mature miRNAs and the hairpins are named -1 and -2. The two copies in Cx. quinquefasciatus are named -1a and -1b because their mature miRNAs differ by one nucleotide. Only the hairpin structure for aae-miR-M1-1 is shown. All five miRNAs shown in panel A have homologs in An. gambiae.

Figure 2

Alignments and stem-loop structures of a novel miRNA cluster within the intron of a gene encoding a transcription factor. See Table 1 for naming and sequence locations of these miRNAs. There are two hairpins for the same miR-N1 (-1 and -2) in both Ae. aegypti and Cx. quinquefasciatus. Only the Ae. aegypti hairpin structures for the miR-N1 pre-miRNAs are shown. Aae-miR-N2 and cqu-miR-N3 are only found in Ae. aegypti and Cx. quinquefasciatus, respectively. Thus there are no alignments for these two miRNAs. Arrows point to the mature miRNA sequences from 5' to 3'. Dashed arrow for cqu-miR-N3 reflects the fact that we do not yet have the direct sequence for this miRNA. The mature cqu-miR-N3 sequence was predicted according to the conserved seed sequence shared with miR-N1 and miR-N2, which was confirmed by northern blots using anti cqu-miR-N3 as a probe (see Figure 7).

Figure 3

Expression patterns of Ae. aegypti homologs of previously known miRNAs. Only Ae. aegypti RNA samples were examined. The top panels are northern results and the bottom panels are RNA gel images for verification of small ribosomal RNA and tRNA integrity and loading of total RNA. Emb, pooled embryos between 0-36 hr after egg deposition; Larvae; mixed instar larvae; Pupae, mixed puape; F, adult females one to five days after emergence; M, adult males one to five days after emergence. 10 μg of total RNA were used per sample.

Figure 4

Higher levels of miRNAs are observed in the female Ae. aegypti midgut 24 hrs after blood feeding (Gut_BF) compared to sugar feeding (Gut_SF). Three-day old females were either fed on blood or sugar and dissected 24 hrs later. 10 μg of total RNA were used per sample. The top panels are northern results and the bottom panels are RNA gel images for verification of small ribosomal RNA and tRNA integrity and loading of total RNA.

Figure 5

Four mosquito-specific miRNAs that are expressed in all four species of three highly divergent genera. MiRNAs examined include miR-M1 (A), miR-1175 (B), miR-1890 (C), and miR-1891 (D). Expression was examined across the developmental stages of An. stephensi, An. gambiae, Ae. aegypti, and T. amboinensis. The top panels are northern results and the bottom panels are RNA gel images for verification of small ribosomal RNA and tRNA integrity and loading of total RNA. Emb, pooled embryos between 0-36 hr after egg deposition; L1+2, pooled 1^st and 2^nd instar larvae; L3+4, pooled 3^rd and 4^th instar larvae; Pupae, mixed pupae; F, adult females one to five days after emergence; M, adult males 1-5 days after emergence. 15 μg of total RNA per sample for An. stephensi, An. gambiae, and Ae. aegypti were used. 10 μg of T. amboinensis total RNA per sample were used. For T. amboinensis northerns, 3^rd instar larvae were not included. For the T. amboinensis miR-M1 northern, hybridization and washes were carried out at 49°C instead of 42°C to reduce background across the membrane.

Figure 6

MiR-1174 is expressed in An. stephensi, An. gambiae, and Ae. aegypti, but not in T. amboinensis. The top panels are northern results and the bottom panels are RNA gel images for verification of small ribosomal RNA and tRNA integrity and loading of total RNA. Emb, pooled embryos between 0-36 hr after egg deposition; L1+2, pooled 1^st and 2^nd instar larvae; L3+4, pooled 3^rd and 4^th instar larvae; Pupae, mixed puape; F, adult females one to five days after emergence; M, adult males one to five days after emergence. 15 μg of total RNA per sample for An. stephensi, An. gambiae, and Ae. aegypti were used. 10 μg of T. amboinensis total RNA per sample were used. For T. amboinensis, 3^rd instar larvae were not included. "+ Cont" indicates a positive control which was An. stephensi embryos (12-24 hr).

Figure 7

MiR-N1, miR-N2, and miR-N3 expression is restricted in particular lineages in mosquitoes. A) miR-N1 is expressed in Ae. aegypti and Cx. quinquefasciatus, but not in An. stephensi nor T. amboinensis. Emb, pooled embryos between 0-36 hr after egg deposition; L1+2, pooled 1^st and 2^nd instar larvae; L3+4, pooled 3^rd and 4^th instar larvae; Pupae, mixed pupae; F, adult females one to five days after emergence; M, adult males one to five days after emergence. Culex Emb, Cx. quinquefasciatus embryos 0-24 hrs after egg deposition. "+ Cont", positive control, Ae. aegypti embryos (12-24 hr). 15 μg of total RNA per sample for An. stephensi and Ae. aegypti were used. 10 μg of T. amboinensis total RNA per sample were used. For T. amboinensis, 3^rd instar larvae were not included. B) miR-N2 is expressed in Ae. aegypti but not detected in Cx. quinquefasciatus and An. stephensi embryos. Symbols are as in A. Anopheles Emb, pooled An. stephensi embryos between 0-36 hr after egg deposition. C) miR-N3 is expressed in Cx. quinquefasciatus, but not detected in Ae. aegypti and An. stephensi embryos. Symbols are as in A and B. Aedes Emb, pooled Aedes aegypti embryos between 0-36 hr after egg deposition.