A New Short Oligonucleotide-Based Strategy for the Precursor-Specific Regulation of microRNA Processing by Dicer

Abstract

The precise regulation of microRNA (miRNA) biogenesis seems to be critically important for the proper functioning of all eukaryotic organisms. Even small changes in the levels of specific miRNAs can initiate pathological processes, including carcinogenesis. Accordingly, there is a great need to develop effective methods for the regulation of miRNA biogenesis and activity. In this study, we focused on the final step of miRNA biogenesis; i.e., miRNA processing by Dicer. To test our hypothesis that RNA molecules can function not only as Dicer substrates but also as Dicer regulators, we previously identified by SELEX a pool of RNA oligomers that bind to human Dicer. We found that certain of these RNA oligomers could selectively inhibit the formation of specific miRNAs. Here, we show that these specific inhibitors can simultaneously bind both Dicer and pre-miRNAs. These bifunctional riboregulators interfere with miRNA maturation by affecting pre-miRNA structure and sequestering Dicer. Based on these observations, we designed a set of short oligomers (12 nucleotides long) that were capable of influencing pre-miRNA processing in vitro, both in reactions involving recombinant human Dicer and in cytosolic extracts. We propose that the same strategy may be used to develop effective and selective regulators to control the production of any miRNA. Overall, our findings indicate that the interactions between pre-miRNAs and other RNAs may form very complex regulatory networks that modulate miRNA biogenesis and consequently gene expression.

How short RNAs impact the human ribonuclease Dicer activity: putative regulatory feedback-loops and other RNA-mediated mechanisms controlling microRNA processing

“…3, demonstrated that hDicer efficiently bound ssRNAs longer than ~20-nt, while it did not form a complex with a 12-nt ssRNA, and binding of a 14-nt ssRNA was very inefficient. These results also confirmed our previous observations that 12-nt ssRNA oligonucleotides, designed to interact with apical and/or single-stranded regions of pre-miRNAs, do not interact with hDicer (Kurzynska-Kokorniak et al, 2013).…”

“…Preparations of hDicer are also commercially available. Our previous studies focusing on the regulation of the hDicer cleavage activity by short RNA molecules were carried out with commercial enzymes (Tyczewska et al, 2011;Kurzynska-Kokorniak et al, 2013). While we found them appropriate for assays based on pre-miRNA cleavage reactions, they do not seem to be suitable for detailed RNA-binding studies.…”

“…The 5′-end labeling of oligonucleotides. The 5′-32 P oligonucleotide labeling by T4 Polynucleotide Kinase (Promega) was performed as described previously (Tyczewska et al, 2011;Kurzynska-Kokorniak et al, 2013). The 32 P-labeled oligonucleotides were purified by polyacrylamide gel electrophoresis (PAGE), in 8% denaturing gels, and resuspended in water to final concentration of approximately 10 000 cpm/µl.…”

“…Our previous studies showed that the activity of hDicer can be affected by short RNA molecules binding to the enzyme, substrate or both (Tyczewska et al, 2011;Kurzynska-Kokorniak et al, 2013;Kurzynska-Kokorniak et al, 2015). In the pool of the investigated ssRNAs there were species ranging from 12-to 62-nt.…”

“…Thereby, other viral transcripts are protected from cleavage. In vitro studies conducted by our group have also indicated that the activity of human Dicer can be influenced by short RNA molecules that either bind to Dicer or interact with its substrates, or both (Tyczewska et al, 2011;Kurzynska-Kokorniak et al, 2013). Those studies were carried out with commercially available Dicers.…”