Abstract
As key regulators of gene expression, microRNAs (miRNAs) have emerged as targets in basic experimentation and therapy. Administration of DNA-encoded RNA molecules, targeting miRNAs through base pairing, is one viable strategy for inhibiting specific miRNAs. A naturally occurring circular RNA (circRNA), ciRS-7, serving as a miRNA-7 (miR-7) sponge was recently identified. This has sparked tremendous interest in adapting circRNAs for suppressing miRNA function. In parallel, we and others have demonstrated efficacy of expressed anti-miRNA Tough Decoy (TuD) hairpins. To compare properties of such inhibitors, we express ciRS-7 and TuD-containing miRNA suppressor transcripts from identical vector formats adapted from RNA polymerase II-directed expression plasmids previously used for production of ciRS-7. In general, markedly higher levels of miR-7 suppression with TuD transcripts relative to ciRS-7 are observed, leading to superior miRNA sponge effects using expressed TuD hairpins. Notably however, we find that individual ciRS-7 transcripts are more potent inhibitors of miR-7 activity than individual TuD7-containing transcripts, although each miR-7 seed match target site in ciRS-7 is, on average, less potent than the perfectly matched target sites in the TuD motif. All together, our studies call for improved means of designing and producing circRNAs for customized miRNA targeting to match TuD hairpins for tailored miRNA suppression.
Highlights
MicroRNAs regulate gene expression at the posttranscriptional level and have emerged as key regulators of almost all biological processes
In accordance with our previous work, the tough decoy (TuD)-7 motif was fused to the 30 end of a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE)-containing eGFP reporter gene,[11,14] whereas circRNA sponge for miR-7 (ciRS-7) was generated by back-splicing of a transcript containing the ciRS-7 region flanked by splice sites and regions known to be required for circle formation
As a control for miR-7 inhibition mediated by ciRS-7, a vector encoding a 62-bp multiple cloning site (MCS) between the splice acceptor (SA) and splice donor sites instead of ciRS-7 was included, whereas an eGFP-WPRE-MCS expression cassette devoid of the TuD motifs was used as control for eGFP-WPRETuD-7 (Figure 1A)
Summary
MicroRNAs (miRNAs) regulate gene expression at the posttranscriptional level and have emerged as key regulators of almost all biological processes. The vector-encoded antagomiRs successfully have been used to suppress the activity of specific miRNAs both in vitro and in vivo,[4,5] the need for more effective miRNA inhibitors has inspired various new designs of vector-encoded miRNA inhibitors.[6,7,8,9,10] One of these, designated the tough decoy (TuD) miRNA sponge, has been shown to be efficient.[8,11] The TuD RNA molecule is approximately 120 nt long and is designed to form a hairpin-shaped structure with stabilizing stems in both top and bottom and an intervening unpaired region consisting of two perfect miRNA target sites (Figure S1).[8] To avoid cleavage of the TuD molecule by Ago-2 upon miRNA binding, each of the miRNA target sites contains a 4-nt long bulge opposite to nucleotide 10–11 of the bound miRNA. We have previously shown high efficiency of RNA pol II-transcribed TuDs fused to a reporter gene and the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE).[11,14] Whereas TuDs are typically exploited for suppression of a specific miRNA species and potentially families of miRNAs, sophisticated new designs allow simultaneous suppression of multiple miRNAs by clustered TuDs15 and alleviation of small hairpin RNA off-target effects using TuDs targeting the passenger strand.[16,17]
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