MicroRNA sponges: progress and possibilities

Abstract

The microRNA (miRNA) "sponge" method was introduced three years ago as a means to create continuous miRNA loss of function in cell lines and transgenic organisms. Sponge RNAs contain complementary binding sites to a miRNA of interest, and are produced from transgenes within cells. As with most miRNA target genes, a sponge's binding sites are specific to the miRNA seed region, which allows them to block a whole family of related miRNAs. This transgenic approach has proven to be a useful tool to probe miRNA functions in a variety of experimental systems. Here we will discuss the ways sponge and related constructs can be optimized and review recent applications of this method with particular emphasis on stable expression in cancer studies and in transgenic animals.

FIGURE 1.

(A) In the absence of sponge treatment, target mRNAs (gray) for a particular miRNA seed family (red complexes) are repressed. Dashed lines indicate mRNA decapping and degradation. (B) After introduction of the sponge transgene, sponge mRNAs (green) are expressed at a high level and sequester the miRNA complexes, rescuing the expression of the endogenous targets. Sponge-treated cells can be identified by their eGFP reporter expression. (C) Pairing of a miRNA with a bulged sponge site shows mismatches opposite miRNA nucleotides 9–12. The miRNA seed region is highlighted.

FIGURE 2.

(A) Tissue-specific expression of the Gal4 transcription factor was used to drive miRNA sponge expression under the control of upstream activating sequences (UAS) in transgenic fruit flies. (B) Dissection of a complex phenotype using tissue-specific sponges. A developmental defect in the axonal branching and synaptic boutons of neuromuscular junctions (NMJ) was observed in the miR-8 knockout (second panel) and in miR-8 heterozygous flies expressing a miR-8 sponge inhibitor specifically in muscle (fourth panel). Wild-type appearance of the NMJ is seen in the miR-8 heterozygote (first panel) and in miR-8 heterozygous flies expressing a miR-8 sponge specifically in neurons (third panel). Sponge expression is indicated by GFP fluorescence (shown in green).