Recent advances in RNA sequencing (RNA-seq) techniques and sequence analysis methods have led to the discovery of thousands of exonic circular RNAs (circRNAs), expressed in many species. These covalently closed RNA circles were assumed to be alternative products of pre mRNA processing by the spliceosome. Being investigated more extensively only during the last five years, few circRNA candidates were characterized, and the understanding of their global biological relevance is still very limited. The currently best investigated circRNA is CDR1as/ciRS 7, which contains more than 70 miR-7 binding sites. It is thought to function as a microRNA (miRNA) sponge by competing for miR 7 binding with its targets. Due to their elevated stability compared with linear RNAs, circRNAs are particularly attractive for biotechnological and therapeutic applications. In order to experimentally substantiate the hypothesis that circRNAs are processed by the spliceosome, plasmid-encoded circRNA splicing reporters (minigenes) were generated. Sequence elements of the natural gene context were gradually removed and a detailed mutational analysis of splice signals was performed. The minigene derivatives were then transfected into cells and processing products were detected via RT-PCR. With this approach, the requirement of fundamental splicing signals for efficient and precise exon circularization was demonstrated. The results provide valid evidence for the involvement of the spliceosome in circRNA biogenesis.The very abundant and liver-specific miR-122 is an essential host factor in hepatitis C virus (HCV) infection. Miravirsen, which sequesters miR 122, is the first locked nucleic acid (LNA)-modified antisense oligonucleotide (oligo) drug for HCV treatment that has entered clinical trials. Based on the concept of the natural CDR1as/ciRS-7 miRNA sponge, artificial circRNA sponges for functional sequestration of miR-122 from HCV RNA were designed. The artificial circRNAs were synthesized in vitro by enzyme-based transcription and ligation of eight consecutive binding sites, followed by gel purification of the circular miRNA sponges. The in vitro generated circRNAs were transfected into cells and analyzed with respect to their subcellular distribution and their stability. Functional inhibition of miR-122 by the circRNAs sponges was tested in three different HCV reporter systems. Efficacy of the miR-122 circRNA sponges was analyzed in comparison to an unspecific circRNA, to their linear counterparts, as well as to miravirsen. The designed circRNA sponges clearly and specifically caused HCV-adverse effects in all tested reporter systems, which is evidence for the functional sequestration of miR 122. This demonstrates the potential of circRNAs to extend the spectrum of RNA therapeutics as pharmaceutical products in the future.
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