microRNAs (miRNAs) constitute a class of small (18-22 nucleotides) non-coding RNAs that bind to the 3’-untranslated region of target messenger RNAs (mRNAs) and have transcriptional or translational silencing functions in eukaryotes. Sequence-specific silencing initiated in one cell can be observed in nearby or faraway cells or even in the whole organism. In multi-cellular organisms intercellular communication is essential for survival and procreation of life. Even though exosomes are known to transport miRNAs, there is little information that addresses how these miRNAs are protected from ribonucleases that are ubiquitous in intracellular and extracellular microenvironments including those inside the intracellular organelles and in exosomal vesicles. Similar to soluble factors, miRNAs mediate cell-to-cell communications and are engaged in a diverse set of biological mechanisms and homeostatic pathways. Previously, we have hypothesized that miRNAs form triplex-forming complexes (TFCs) that become resistant to nucleases. Here, we hypothesize that miRNAs form triplex complexes in order to survive the digestive effects of endonucleases, ubiquitous in all types of extracellular and intracellular environments. In order to evaluate our hypothesis we utilized two strategies: first, we treated HeLa cells with various endonucleases (i.e. Rnase, Dnase) and second, we blocked the biosynthesis of miRNAs. We determined the presence of TFC by treating cells with a variety of single and double-stranded endo/exonucleases. By employing anti-TF-monoclonal antibodies, we demonstrate that miRNAs utilize a TFC that is resistant to various RNA and DNA nucleases. The confirmation of involvement of miRNA in TFC was further corroborated by the blocking of miRNA biosynthesis by using two polymerase II (Pol II) inhibitors: α-Amanitin, and Pol III inhibitor, Tagetin. Time-course analyses of Pol II/III inhibitors exhibited a gradual decrease in TFCs after 24, 48 and 72 hrs of treatment, with elimination of TFCs after 72 hrs. The role of TFC was further explored by transfecting HeLa cell lines with two miRNAs (miR-let-7i and miR-195) and staining the transiently transfected cells for TFC antibodies, which showed TFC transmission via long-micropathways. Transportation of TFCs by means of cell-cell communication (i.e. micropathways) and exosomes was also demonstrated. Our findings may have important implications in miRNA research related to the delivery of therapeutic miRNAs in malignancy, infectious diseases and other immunologic illnesses.
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