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. 2018 Oct 10;38(9):1410–1431. doi: 10.1038/s41388-018-0530-7

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a–d AGO2 acetylation enhances its binding with pre-miR-19b1. a Serum stimulates pre-miR-19b1 binding to AGO2. 293T cells were co-transfected pre-miR-19b1 with Flag-AGO2 for 20 h, then were serum-starved for 24 h, and followed by stimulation with 20% serum for indicated times. Then cells were lysed by RIP lysis buffer for the RIP assay with anti-Flag antibody, followed by northern blotting analysis. b P300/CBP enhances the interaction between pre-miR-19b1 and AGO2. Pre-miR-19b1 and Flag-tagged AGO2 were co-transfected with HA-P300 or HA-CBP into 293T cells, respectively. The RIP/northern blotting analysis were performed as before. c HDAC7 but not HDAC6 reduces the interaction between pre-miR-19b1 and AGO2. HA-tagged AGO2 and pre-miR-19b1 were co-transfected with Flag-HDAC6 or Flag-HDAC7 into 293T cells. The RIP/northern blotting analysis were performed as before. d Acetyl-mutants K493R or K720R, but not K355R of AGO2 decreases its interaction with pre-miR-19b1. AGO2-WT and mutants AGO2-K355R, -K493R, -K720R and -3KR were individually co-transfected with pre-miR-19b1 into 293T cells. 48 h later, The RIP/northern blotting analysis was performed as before. The efficiencies of RNA immunoprecipitation and mature miR-19b in Input were determined by WB Northern blotting, respectively, in all above experiments. e–h The UGUGUG Motif in the Terminal Loop of pre-miR-19b1 is a specific processing feature for the interaction with acetylated AGO2. e, f Deletion or mutation of the UGUGUG motif in the terminal loop of pre-miR-19b1 impairs pre-miR-19b1 processing mediated by AGO2 in a DICER dependent manner. e pre-miR-19b1-WT, pre-miR-19b1-∆UGUGUG or pre-miR-19b1-AUAUAU was co-transfected with Myc-AGO2 into 293T-shControl or 293T-shDICER cells; (f) pre-miR-19b1-WT, pre-miR-19b1-∆UG, pre-miR-19b1-∆UGUG, pre-miR-19b1-UGAUUG or pre-miR-19b1-AUAUUG were co-transfected with Myc-AGO2 into 293T cells, 48 h later, the total RNAs were extracted and followed by northern blotting analysis. g The UGUGUG motif of pre-miR-19b1 is necessary for its interaction with acetylated AGO2. Pre-miR-19b1-WT, pre-miR-19b1-UGAUUG, pre-miR-19b1-AUAUUG and pre-miR-19b1-AUAUAU were co-transfected with Flag-AGO2-WT or -3KR into 293T cells. 48 h later, cells were lysed by for the RIP assay with anti-Flag antibody, the interaction of pre-miR-19b1 with Flag-AGO2-WT or Flag-AGO2-3KR was detected by northern blotting analysis. h Serum stimulation enhances the association of AGO2 with pre-miR-19b1-WT, but not the UGUGUG-motif mutated pre-miR-19b1. Pre-miR-19b1-WT, pre-miR-19b1-UGAUUG, pre-miR-19b1-AUAUUG or pre-miR-19b1-AUAUAU were co-transfected with Flag-AGO2 into 293T cells for 20 h, then were serum-starved for 24 h, and followed by stimulation with 20% serum for for 3 h. The RIP/northern blotting analysis was performed as before. The efficiencies of RNA immunoprecipitation and mature miR-19b in input were determined by WB and Northern blotting, respectively

Fig. 5 — a–d AGO2 acetylation enhances its binding with pre-miR-19b1. a Serum stimulates pre-miR-19b1 binding to AGO2. 293T cells were co-transfected pre-miR-19b1 with Flag-AGO2 for 20 h, then were serum-starved for 24 h, and followed by stimulation with 20% serum for indicated times. Then cells were lysed by RIP lysis buffer for the RIP assay with anti-Flag antibody, followed by northern blotting analysis. b P300/CBP enhances the interaction between pre-miR-19b1 and AGO2. Pre-miR-19b1 and Flag-tagged AGO2 were co-transfected with HA-P300 or HA-CBP into 293T cells, respectively. The RIP/northern blotting analysis were performed as before. c HDAC7 but not HDAC6 reduces the interaction between pre-miR-19b1 and AGO2. HA-tagged AGO2 and pre-miR-19b1 were co-transfected with Flag-HDAC6 or Flag-HDAC7 into 293T cells. The RIP/northern blotting analysis were performed as before. d Acetyl-mutants K493R or K720R, but not K355R of AGO2 decreases its interaction with pre-miR-19b1. AGO2-WT and mutants AGO2-K355R, -K493R, -K720R and -3KR were individually co-transfected with pre-miR-19b1 into 293T cells. 48 h later, The RIP/northern blotting analysis was performed as before. The efficiencies of RNA immunoprecipitation and mature miR-19b in Input were determined by WB Northern blotting, respectively, in all above experiments. e–h The UGUGUG Motif in the Terminal Loop of pre-miR-19b1 is a specific processing feature for the interaction with acetylated AGO2. e, f Deletion or mutation of the UGUGUG motif in the terminal loop of pre-miR-19b1 impairs pre-miR-19b1 processing mediated by AGO2 in a DICER dependent manner. e pre-miR-19b1-WT, pre-miR-19b1-∆UGUGUG or pre-miR-19b1-AUAUAU was co-transfected with Myc-AGO2 into 293T-shControl or 293T-shDICER cells; (f) pre-miR-19b1-WT, pre-miR-19b1-∆UG, pre-miR-19b1-∆UGUG, pre-miR-19b1-UGAUUG or pre-miR-19b1-AUAUUG were co-transfected with Myc-AGO2 into 293T cells, 48 h later, the total RNAs were extracted and followed by northern blotting analysis. g The UGUGUG motif of pre-miR-19b1 is necessary for its interaction with acetylated AGO2. Pre-miR-19b1-WT, pre-miR-19b1-UGAUUG, pre-miR-19b1-AUAUUG and pre-miR-19b1-AUAUAU were co-transfected with Flag-AGO2-WT or -3KR into 293T cells. 48 h later, cells were lysed by for the RIP assay with anti-Flag antibody, the interaction of pre-miR-19b1 with Flag-AGO2-WT or Flag-AGO2-3KR was detected by northern blotting analysis. h Serum stimulation enhances the association of AGO2 with pre-miR-19b1-WT, but not the UGUGUG-motif mutated pre-miR-19b1. Pre-miR-19b1-WT, pre-miR-19b1-UGAUUG, pre-miR-19b1-AUAUUG or pre-miR-19b1-AUAUAU were co-transfected with Flag-AGO2 into 293T cells for 20 h, then were serum-starved for 24 h, and followed by stimulation with 20% serum for for 3 h. The RIP/northern blotting analysis was performed as before. The efficiencies of RNA immunoprecipitation and mature miR-19b in input were determined by WB and Northern blotting, respectively