Abstract
Small-interfering RNAs and microRNAs are small ∼21–22 nucleotide long RNAs capable of posttranscriptional suppression of gene expression. The synthetic siRNAs are especially designed to target pre-specified genes and are common molecular biology tools. The miRNAs are endogenous regulators of gene expression found in a wide variety of eukaryotes. miRNAs are currently utilized for diagnostics applications. Therapeutically, various miRNA-antagonizing tools are being explored and miRNAs are also utilized for cell-specific inhibition of the expression of gene therapy vectors harboring target sites for specific miRNAs. Here we show, for the first time, that siRNAs and miRNAs can be harnessed to induce gene expression. We designed special expression vectors in which target sites for artificial siRNAs or endogenous miRNAs are located between the transgene and an Upstream Inhibitory Region (UIR). We hypothesized that cleavage of the mRNA by siRNAs or miRNAs will separate the transgene from the UIR and the resulting uncapped mRNA will be capable of being translated. A UIR composed of seven open reading frames was found to be the most efficient inhibitor of the translation of the downstream transgene. We show that under such a configuration both artificial siRNAs and endogenous miRNAs were capable of inducing transgene expression. We show that using the diphtheria toxin A-chain gene, in combination with target sites for highly expressed miRNAs, specific induction of cell-death can be achieved, setting the stage for application to cancer therapy.
Highlights
The discovery of RNA interference [1] led to the uncovering of a basic cellular regulatory mechanism common to a wide spectrum of organisms
We provide proof that this unique construct architecture leads to induction of gene expression by both synthetic small interfering RNAs (siRNAs) and endogenous miRNAs in a wide variety of human cell-lines
We reasoned that for achieving induction of gene expression by siRNA or miRNA we would need a large number of mRNA molecules of the transgene
Summary
The discovery of RNA interference [1] led to the uncovering of a basic cellular regulatory mechanism common to a wide spectrum of organisms. RNA interference by long double-stranded (ds) RNAs turned out to be driven by small ,21 nucleotides long RNA molecules derived from the long dsRNA [3, 4] These small interfering RNAs (siRNAs) were shown to function in mammalian cells as potent and specific inhibitors of gene expression and quickly became an important tool both experimentally and therapeutically. MicroRNAs (miRNAs) were first discovered in 1993 [5] but their wide evolutionary abundance and regulatory roles were established at the beginning of our century [6] These are ,21–22 nucleotide long RNA molecules, derived from hairpin structure embedded in long mRNAs or introns of various genes. Perfect or near perfect pairing of the miRNA to the mRNA target site leads to Argonaute-catalysed mRNA cleavage [8,9,10]
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