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. 2020 Nov;26(11):1603–1620. doi: 10.1261/rna.075309.120

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© 2020 Sun et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 6. — MIR222HG forms RNP complex containing ILF3/2 protein and other RNAs. (A) In vitro biotin-RNA pull-down followed by immunoblotting to detect the interaction between MIR222HG and ILF3/ILF2. YFP RNA is used as negative RNA control. α-Tubulin is used as negative protein control. (B) ILF3 RIP followed by RT-qPCR to quantify the interaction between ILF3 and MIR222HG or EIF4E (3′-UTR region) or DNM3OS lncRNA in WI-38 cells. GAPDH is used as negative control. (C) In vitro biotin-RNA pull-down followed by RT-qPCR to determine the interaction between MIR222HG and ILF3/2-target RNAs in WI-38 cells. Relative fold enrichment of EIF4E mRNA and DNM3OS lncRNA are calculated by comparing to biotin-YFP. GAPDH is used as negative control. (D) RT-qPCR to quantify the levels of DNM3OS lncRNA in control and MIR222HG-depleted WI-38 cells in quiescent and 6-h post-serum-stimulated WI-38 cells. (E) RT-qPCR to quantify the levels of DNM3OS in control and ILF3-depleted WI-38 cells post 6-h serum-stimulation. (F) RNA stability analyses of DNM3OS in control and MIR222HG-depleted WI-38 cells. RNA is extracted from cells incubated with actinomycin D for indicated time points (min). Relative levels of RNA are measured via RT-qPCR. (G) In vitro biotin-RNA pull-down followed by RT-qPCR to determine the interaction between MIR222HG and DNM3OS in control and ILF3-depleted serum-stimulated WI-38 cells. Relative fold enrichment of DNM3OS lncRNA is calculated by comparing to biotin-YFP. GAPDH is used as negative control. (H) Schematic of ILF3/2-MIR222HG-DNM3OS RNP complex. (*) P ≤ 0.05, (**) P ≤ 0.01, (***) P ≤ 0.001, (****) P ≤ 0.0001 by two-tailed Student's t-test, n = 3 for all figures. Error bars represent standard deviation.