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. Author manuscript; available in PMC: 2010 Oct 9.
Published in final edited form as: Mol Cell. 2009 Oct 9;36(1):61–74. doi: 10.1016/j.molcel.2009.08.008

Figure 2. miR-214 Down-Regulates Ezh2 by Direct Targeting Its 3′UTR.

Figure 2

(A) Schematic representation of luciferase reporter constructs. The predicted structure of each base-paired Ezh2 3′ UTR/wild-type or mutated miRNA hybrid is diagrammed, and the calculated free energy (ΔG) in kilocalories per mole of the 5′ seed region of each hybrid is shown on the right. The top strand in each diagram represents 5′ to 3′ Ezh2 3′ UTR WT and mutants (nucleotides in color), and the bottom strand represents the microRNAs. (B) Luciferase reporters shown in (A) were transfected into C2C12 myoblasts (50%, 100% confluent, or differentiated in DM for 24 hrs) and luciferase activity was determined 48 hrs after transfection. The ratio of reporter (Firefly Luciferase, FL) to control plasmid (Renilla Luciferase, RL) in relative luminescence units was normalized for each reporter to the buffer control and plotted as fold increase or decrease of the control value. Error bars represent standard deviations from 3 independent experiments done in triplicate (*** p<0.0005; ** p<0.005). (C) Control luciferase reporter (pGL3), pGL3 with wild-type Ezh2 3′UTR (WT), or with Ezh2 3′ UTR mutated miR-214 site (Mut) were transfected into C2C12 cells overexpressing empty vector (left panel), wild-type miR-214 (miR-214wt, middle panel), or mutated miR-214 (miR-214-mut, right panel). The sequences of miR-214-wt and the mutations introduced into miR-214-mut are indicated on the bottom. Luciferase activity was determined as described in (B). *** p<0.0005; ** p<0.005. (D) C2C12 cells were co-transfected with either control or anti-miR-214 oligomers, pGL3 (control vector), pGL3-Ezh2 3′ UTR (WT), or pGL3-Ezh2 3′ UTR mutated miR-214 site (Mut) reporter constructs. Luciferase activity was determined as described in (B). **** p<10-6. (E) Schematic representation of RNA-immnuoprecipitation (upper panel) and miRNA pull-down experiments (lower panel). (F) Cell extracts were prepared from 24 hours differentiated C2C12 cells overexpressing empty vector, wild-type miR-214, mutated miR-214 or miR-22 and immnuoprecipitated with either Ago2 antibody or IgG and blotted with Ago2 antibody (top panel). Asterisk indicates 55 kDa IgG heavy chains. The precipitated RNA was converted to cDNA and amplified by real-time PCR using Ezh2 primers (lower panel). Error bars represent standard deviations (n=3), * p< 0.05, ** p< 0.005. (G) C2C12 cell extracts were prepared from cells transfected with biotinylated double-strand RNA miR-214 or miR-214 mutant and subjected to either immunoblot (left panel) or pull-down assay with streptavidin beads. The pulled-down RNA was converted to cDNA and amplified by real-time PCR using Ezh2 or SIRT1 primers. Error bars represent standard deviations from 3 independent experiments. * p < 0.05, ** p< 0.005. (H) Cell extracts from C2C12 cells transfected with control (pGL3), WT Ezh2 3′UTR (WT), or 214Mut Ezh2 3′ UTR (Mut) luciferase constructs were immunoprecipitated with either Ago2 antibody or unrelated IgG. The precipitated RNA was converted to cDNA and amplified by real-time PCR using primers spanning the luciferase and Ezh2 3′UTR junction. The values were corrected for the amount of total luciferase –Ezh2 3′ UTR RNAs (either WT or Mut), measured by real-time PCR. Error bars represent standard deviations from 3 independent experiments, * p< 0.05.