Supplementary MaterialsSupplementary information, Figure S1: Onconase downregulates miR-155 and miR-21 and upregulates their targets in mesothelioma cells. and miR-21 precursor; lanes 4-6, Dicer, miR-21 precursor, and raising levels of onconase (0.1, 0.5, and 2.5?M); street 7, oncoanse (0.1?M) and miR-21 precursor. (D-F) Primer expansion analyses from the cleavage sites of onconase in miR-155 precursor (D), miR-21 precursor (E), and miR-29a precursor (F). Lanes 1-4, DNA sequencing of miRNA precursors; lanes 5-7, dedication from the positions of cleavage with 1.0?M and 0.1?M onconase, and without onconase, respectively. Arrows reveal positions of cleavages. The sequences which were identified by primers in both of DNA sequencing and primer expansion assays are demonstrated in red. Considering that onconase ideally cleaves miRNA precursors (Shape 1A and ?and1B)1B) which miRNA precursors are processed into miRNAs by Dicer in cells, we speculated that onconase may reduce miRNA Rabbit Polyclonal to VAV3 (phospho-Tyr173) expression through troubling Dicer-mediated adult miRNA production in tumor cells. Certainly, using Dicer digesting analyses (Supplementary info, Data S1), we discovered that digesting of pre-miR-21 into adult miR-21 by Dicer was dose-dependently decreased by onconase (Shape 1C). These outcomes claim that onconase degrades miRNA precursors and consequently leads to less creation of mature miRNAs made by Dicer. Finally, we BIIB021 mapped the cleavage sites of onconase in miRNA precursors. Using primer expansion analyses (Supplementary info, Data S1), we discovered that pre-miR-155 was primarily cleaved at C39-U40 and U40-G41 (Shape 1D), pre-miR-21 at U21-G22, U31-G32, U34-G35, and U43-G44 (Shape 1E), and pre-miR-29a at U14-U15 and U18-G19 (Shape 1F). These total outcomes indicate that onconase seems to mainly cleave miRNA precursors at UG and UU residues, which act like the cleavage specificity of onconase seen in BIIB021 tRNAs17. In conclusion, our outcomes indicate that onconase downregulates miRNA expression in mesothelioma cells ubiquitously. Interestingly, our biochemical assays reveal that onconase preferentially degrades miRNA precursors and mildly affects mature forms of BIIB021 miRNAs. Given that miRNA precursors resemble tRNAs, the well-documented downstream RNA species of onconase, with 70-nt long and hairpin structure, we speculate that an appropriate secondary structure might be required for onconase substrates. Indeed, similar to the predominant cleavages of onconase in tRNAs located in the variable loop or stem of D-arm17, we found that the cleavage sites of onconase in miRNA precursors are mapped at both loop and stem regions. Given that oncogenic miRNAs are upregulated in cancer cells while tumor suppressive miRNAs are often downregulated18, it is likely that onconase exerts its antitumor activity through targeting miRNAs, i.e., mainly oncogenic miRNAs. Taken together, our study reveals miRNA precursors as a novel class of RNA targets for onconase in addition to tRNAs, bringing new insights into the mechanisms of onconase-mediated cytotoxicity in cancer cells. Acknowledgments We thank Prof BIIB021 En-Duo Wang (Institute of Biochemistry and Cell Biology, SIBS, CAS) for providing human tRNAArg. This work was supported by grants from the Ministry of Science and Technology of China (2011CB811303, 2012CB910802, 2011CB966304) and the National Natural Science Foundation of China (30970621, 31170754). Footnotes (Supplementary information is linked to the online version of the paper on the website.) Supplementary Material Supplementary information, Figure S1Onconase downregulates miR-155 and miR-21 and upregulates their targets in mesothelioma cells. Click here for additional data file.(187K, pdf) Supplementary information, Figure S2Onconase ubiquitously downregulates miRNAs with high abundance in mesothelioma cells. Click here for additional data file.(145K, pdf) Supplementary information, Data S1Materials and Methods Click here for additional data file.(78K, pdf).