Insect microRNAs: biogenesis, expression profiling and biological functions

Abstract

MicroRNAs (miRNA) are a class of endogenous regulatory RNA molecules 21-24 nucleotides in length that modulate gene expression at the post-transcriptional level via base pairing to target sites within messenger RNAs (mRNA). Typically, the miRNA "seed sequence" (nucleotides 2-8 at the 5' end) binds complementary seed match sites within the 3' untranslated region of mRNAs, resulting in either translational inhibition or mRNA degradation. MicroRNAs were first discovered in Caenorhabditis elegans and were shown to be involved in the timed regulation of developmental events. Since their discovery in the 1990s, thousands of potential miRNAs have since been identified in various organisms through small RNA cloning methods and/or computational prediction, and have been shown to play functionally important roles of gene regulation in invertebrates, vertebrates, plants, fungi and viruses. Numerous functions of miRNAs identified in Drosophila melanogaster have demonstrated a great significance of these regulatory molecules. However, elucidation of miRNA roles in non-drosophilid insects presents a challenging and important task.

Fig. 1

A model for microRNA biogenesis. MicroRNA loci are typically transcribed by RNA polymerase II (pol II). The transcripts fold into a hair-loop structure known as the pri-miRNA, which is processed in the nucleus by the Drosha/Pasha microprocessor complex to form a ~70nt pre-miRNA. The pre-miRNA is exported to the cytoplasm in a RanGTP/Exp-5 dependent manner for further processing. Within the cytoplasm the pre-miRNA is processed by Dicer-1 (Dcr-1) and Loquacious (Loq) to form the miRNA-miRNA* duplex. The duplex strands are then sorted and miRNA strand is loaded into the RISC complex that typically includes Argonaut 1 (Ago-1). In some instances, the miRNA within RISC undergoes further trimming by Nibbler (Nbr). The miRNA “seed sequence” (nucleotides 2-8 at the 5′ end) binds complementary seed match sites within the 3′ untranslated region of mRNAs, resulting in either translational inhibition or mRNA degradation.

Fig. 2

MicroRNA inactivation via a cholesterol (chl) conjugated oligoribonucleotide (Antagomir, Ant). Typically, the miRNA-miRNA* duplex is incorporated into RISC leading to translational inhibition or mRNA degradation. However, the sequence-specific Ants competitively bind mature miRNAs in RISC thereby preventing miRNAs from binding their target mRNAs. As a result, a given mRNA, destined for translational inhibition or degradation, generates a protein in an incorrect spatiotemporal manner creating undesirable effect in an organism.

Fig. 3

The microRNA sponge (miR-SP) transgenic loss-of-function approach utilizing the GAL4-UAS system in Drosophila. (a) miR-SP design. Ten miRNA binding sites (red) containing mismatches at positions 9-12 were inserted downstream of EGFP (green) in a UAS-containing P-element vector. (b) The resulting transgenic Drosophila line can be crossed to specific Gal4 lines to achieve hybrid transgenic lines with spatiotemporal inhibition of a miRNA. (From Loya et al., 2009, with permission).

Fig. 4

An in vitro cell transfection assay for testing putative miRNA target genes. A 3′ UTR reporter vector harboring a reporter gene and the 3′ UTR of the putative miRNA target gene is co-transfected into an insect cell line with a miRNA mimic of interest. Successful binding of the miRNA mimic to the putative miRNA target will result in the suppression of reporter gene expression.

Fig. 5

The highly conserved miRNA, miR-8 has implicated roles in the insulin signaling pathway in the Drosophila larval fat body. (a) Both male and female miR-8 null flies have a smaller body size compared their wild-type counterparts. (b) Average weight of wild-type (male, n = 65; female, n = 70) and miR-8 null (male, n = 60; female, n = 40) flies. (Hyun et al., 2009, with permission).

Fig. 6

In Drosophila, miR-34 modulates age-associated processes. Loss of miR-34 results in accelerated brain aging, late-onset brain degeneration and a decline in survival. Top left panel, miR-34 mutant flies have normal brain morphology at 3d. Major anatomical structures: CB (central brain), Lo (lobula), LoP (lobula plate), Me (medulla), La (lamina) and Rt (retina). At 3d, control flies have normal brain morphology (not shown), but develop a small number of sporadic vacuoles at 30d (top right panel, arrowheads). Middle panel, aged miR-34 mutants (30d) show striking vacuoles in the medulla (arrows) and other regions of the brain (arrowheads). Bottom, the number of vacuoles in miR-34 mutants is significantly higher than in controls (22.2± 1.8 vs 1.5±0.3 in medulla; 19.2±2.5 vs 7.0±0.9 in other regions of the brain; **=p < 0.001, one-way analysis of variance, with post test: Tukey’s multiple comparison test). Mean ± s.e.m., n=10 independent male fly brains. Scale bar: 0.1mm. (Liu et al., 2012a, with permission).

Fig. 7

Antagomir depletion of miR-275 drastically affects the ability of the Aedes aegypti female mosquito to digest blood. Mosquitoes were injected with either miR-275 antagomir (miR-275-ant) or missense oligonucleotide antagomir probe (ms-ant) at 1 day post-eclosion. Digestive systems, containing the crop (Cr), and anterior and posterior midguts (mg), were dissected 24 h after blood feeding and compared with untreated control (Wild Type). Digestive systems from miR-275-ant treated mosquitoes were full of red-colored (undigested) blood that was mostly located in a largely extended crop. Midguts from ms-ant mosquitoes were similar to those from wild type controls with a black bolus of digested blood. (From Bryant et al. 2010, with permission).